Assessment of mtDNA damage by quantitative PCR . HEK293 cells infected with control ( empty vector ) or shATG5 for 5 days . Cells were then incubated with DMSO or 4 mM 3 - NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h . Cells with or without washout were used for the extraction of total DNA . All DNA samples were used for amplification of 8 . 9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment . PCR products were quantitated by PicoGreen staining using Micro Plate Reader . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . The data in ( A ) were further calculated for the frequency of mtDNA damage . The equation was seen in " Materials and Methods " . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.


Representative images show 8 - oxo - dG staining . Control or shATG5 HeLa cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and then either fixed immediately or washed with fresh medium and incubated for another 1 h . Cells were immunostained with DAPI and anti - 8 - oxo - dG antibody and analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in ( C ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.


Control or shATG5 HEK293 cells stably expressing mito - Keima . Control cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and shATG5 cells were treated with 200 µM H2O2 as a negative control . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Right panels show the pixel intensity of red ( mitochondria within lysosomes ) and green ( mitochondria in the cytoplasm ) from a line . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( E ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.


HeLa cells expressing GFP - LC3 were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were stained with anti - Tom20 and anti - DNA antibodies , then analyzed by confocal microscopy . The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA / Tom20 . Scale bar , 10 µm . Quantification of the LC3 punta colocalizing or contacting with mtDNA / Tom20 in a cell . n = 30 cells from 3 coverslips , Data are presented as mean ± SD ( n = 30 ) , statistical significance was assessed by a one - way ANOVA with Tukey ' s multiple comparisons test , * * * p < 0 . 001 .

O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.


Screening of mitophagy regulators for removing damaged mtDNA . HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors . Five days later , cells were treated with 200 µM H2O2 for 2 h . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima . ShATG5 was used as a negative control . Right panels for each image show the FACS - based mito - Keima dot plots . The y - axis represents the fluorescence emission of mito - Keima at pH 4 . 0 ( lysosome ) , while the x - axis indicates mito - Keima at pH 7 . 0 ( mitochondria ) . The percentages of cells within the different regions are indicated . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.


Control or ATAD3B KO HeLa cells stably expressing mito - Keima were treated with 4 mM 3 - NPA for 2 h and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( B ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


Control or KO ATAD3B HeLa cells were treated with 4 mM 3 - NPA for 2 h . Cells were washed with fresh medium and incubated for another 1 h . Cells were then fixed and immunostained with DAPI and anti - 8 - oxo - dG antibodies and were analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in cells described in ( D ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.


PINK1 KO HeLa cells stably expressing mito - Keima were infected with control or shATAD3B . Five days later , cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were then analyzed and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561nm / 458nm ) of the cells described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


HeLa cells were treated with DMSO , 4 mM 3 - NPA , 8 mM 3 - NPA , or 10 µM CCCP for 2 h , and cell lysates were then analyzed by Western blotting using the indicated antibodies .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.


PINK1 KO HeLa cells stably expressing ATAD3A - Flag ( 3A - Flag ) or ATAD3B - Flag ( 3B - Flag ) were mixed with control PINK1 KO cells respectively , and incubated for 24 h . Cells were treated with 4 mM 3 - NPA ( D ) or 200 µM H2O2 ( F ) for 2 h , and then washed with fresh medium and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and anti - Flag antibodies and analyzed by confocal microscopy . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( E ) or H2O2 ( G ) was quantified by ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by individual two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . Scale bar , 10 µm .

O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.


Control ( GFP + ) and shATAD3B ( GFP - ) PINK1 KO HeLa cells were mixed and incubated for 24 h . Cells were then treated with 4 mM 3 - NPA ( H ) or 200 µM H2O2 ( J ) for 2 h , and washed with fresh medium , and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and DAPI , then analyzed by confocal microscopy . Control or shATAD3B cells are circled by white dashed lines . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( I ) or H2O2 ( K ) was quantified using ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , * * p < 0 . 01 . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.


293T cells were treated with H2O2 for 2 h , and cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with rabbit IgG ( control ) or anti - ATAD3 antibody , followed by Western blotting with anti - LC3B or anti - ATAD3 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O

293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.


ATAD3 DKO HeLa cells expressing GFP - LC3B were transiently transfected with control , ATAD3A - Flag or ATAD3B - Flag . 48 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - Flag M2 affinity gel , and analyzed by Western blotting using anti - Flag or anti - GFP antibodies .

O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ATAD3 DKO HeLa cells expressing GFP-LC3B were transiently transfected with control, ATAD3A-Flag or ATAD3B-Flag. 48 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, and analyzed by Western blotting using anti-Flag or anti-GFP antibodies.


HeLa cells were treated with 200 µM H2O2 for 2 h , and cell lysates were incubated with the indicated GST - ATAD3A or GST - ATAD3B protein fragments ( expressed in E . coli ) coupled to glutathione agarose beads ( Pierce ) for GST pull - down assay . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


The indicated GST - ATAD3A or GST - ATAD3B protein fragments and His - LC3B were expressed in E . coli and purified . GST pull - down assay was performed using glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using anti - GST or anti - His antibodies .

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.


The indicated GST - ATAD3B ( WT ) or GST - ATAD3B ( LIR mutations ) and His - LC3 were expressed in E . coli and purified . GST pull - down assay was performed using the glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


HeLa expressing GFP - LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B . Five days later , cells were treated with DMSO , or 200 µM H2O2 for 2 h . Cells were fixed and immunostained with anti - Tom20 and imaged by confocal microscopy . The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 ( mitochondria ) . Scale bar , 10 µm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O

HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.


Quantification of the GFP - LC3 puncta colocalized or contacted with Tom20 ( mitochondria ) in cells described in ( G ) . Data are presented as mean ± SD ( n = 30 cells from 3 coverslips ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * * p < 0 . 001 .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the GFP-LC3 puncta colocalized or contacted with Tom20 (mitochondria) in cells described in (G). Data are presented as mean ± SD (n =30 cells from 3 coverslips), statistical significance was assessed by a two-way ANOVA, N.S., not significant, ***p < 0.001.


293T cells transiently transfected with control or ATAD3B - Flag were treated with or without 200 µM H2O2 for 2 h . Cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with anti - Flag antibody , followed by Western blotting with anti - Flag or anti - LC3B antibodies . Relative protein levels of LC3B and ATAD3B - Flag were further evaluated by

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

293T cells transiently transfected with control or ATAD3B-Flag were treated with or without 200 µM H2O2 for 2 h. Cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with anti-Flag antibody, followed by Western blotting with anti-Flag or anti-LC3B antibodies. Relative protein levels of LC3B and ATAD3B-Flag were further evaluated by densitometry


Mic10 KO COS7 cells stably expressing ATAD3A - Flag or ATAD3B - Flag were treated with DMSO or 200 µM H2O2 for 2 h , and then fixed and immunostained with anti - Tom20 and anti - Flag antibodies , and analyzed by confocal microscopy . Scale bar , 10 µm .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Mic10 KO COS7 cells stably expressing ATAD3A-Flag or ATAD3B-Flag were treated with DMSO or 200 µM H2O2 for 2 h, and then fixed and immunostained with anti-Tom20 and anti-Flag antibodies, and analyzed by confocal microscopy. Scale bar, 10 µm.


293T cells treated with DMSO ( B ) or H2O2 ( 200 µM , 2 h ) ( D ) , were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) . Relative protein levels of proteins in ( B ) or ( D ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from DMSO - or H2O2 - treated cells were shown ( C and E ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

293T cells treated with DMSO (B) or H2O2 (200 µM, 2 h) (D), were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix). Relative protein levels of proteins in (B) or (D) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from DMSO- or H2O2-treated cells were shown (C and E). Data are presented as mean ± SD (n = 3 independent experiments).


Control , ATAD3A KO , ATAD3B KO , or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min , and then were analyzed by Western blotting with the indicated antibodies .

O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

Control, ATAD3A KO, ATAD3B KO, or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO , 200 µM H2O2 , 10 mM NAC , or 200 µM H2O2 plus 10 mM NAC for 2 h . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies . Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in ( F ) . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

293T cells were treated with DMSO, 200 µM H2O2, 10 mM NAC, or 200 µM H2O2 plus 10 mM NAC for 2 h. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies. Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in (F). Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


143B , 143B ρ0 cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

143B, 143B ρ0 cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO or ddC ( 50 µM ) plus EB ( 0 . 5 µg / ml ) for 10 days . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

293T cells were treated with DMSO or ddC (50 µM) plus EB (0.5 µg/ml) for 10 days. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37℃, and then were analyzed by Western blotting with the indicated antibodies.


ATAD3A KO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) ( K ) . Relative protein levels of proteins in ( K ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown ( L ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

ATAD3A KO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix) (K). Relative protein levels of proteins in (K) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown (L). Data are presented as mean ± SD (n = 3 independent experiments).


ρ0206 _ A and ρ0206 _ B cells were stained with mitoSOX and analyzed by confocal microscopy . DIC , Differential Interference Contrast . Scale bar , 25 µm . Quantification of the MitoSOX fluorescence intensity described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by student ' s t - test , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.


Mitochondria carrying the 3243A > G mutation in the tRNA Leu ( UUR ) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206 _ A ( 100 % wild - type mtDNA ) and ρ0206 _ B ( ~ 90 % m . 3243A > G mtDNA ) cell lines . ρ0206 _ A and ρ0206 _ B cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


ρ0206 _ A , ρ0206 _ B , and ρ0206 - B cells pretreated with ddC ( 50µM ) plus EB ( 0 . 5µg / ml ) for 10 days , and then harvested and lysed . Cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - GAPDH antibodies .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.


ρ0206 _ B cells were infected with control , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA from cells was isolated and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ρ0206_B cells were infected with control, ATAD3B-Flag or ATAD3B (mLIR)-Flag. After two weeks, total DNA from cells was isolated and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


ρ0206 _ B cells were infected with control , ATAD3A - Flag , ATAD3B - Flag , or ATAD3B ( mLIR ) - Flag . After two weeks , the cellular ATP levels were measured . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


MELAS patient fibroblasts were infected with control , ATAD3A - Flag , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MELAS patient fibroblasts were infected with control, ATAD3A-Flag, ATAD3B-Flag or ATAD3B(mLIR)-Flag. After two weeks, total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, **p < 0.01.


Comparison of bacterial colony forming unit ( CFU ) in feces from vehicle - and ABX - treated mice ( n = 5 mice / group ) . n . d . , non - detectable .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.


Representative image and measurements of lengths for CD31 + / LYVE - 1 + lacteal , CD31 + / LYVE - 1 - capillary plexus and DAPI + villus .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.


Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of vehicle - or ABX - treated mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).


Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( F ) presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


Images and comparisons of the VEGFR3 expression ( G ) , presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of the VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


Images and comparison of VE - cadherin LEC junctions in lacteals of jejunum between vehicle - and ABX - treated mice . Each boxed portion is highly magnified for visualization of detailed distributions of VE - cadherin to distinguish button - like ( red arrowheads ) , zipper - like ( open arrowheads ) and mixed LEC junctions in lacteals ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 50 μm .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.


Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals . Asterisks indicate the site in lamina propria where fluorescence intensity ( FI ) was measured . Relative FI divided by mean FI of controls at 16 min ( left ) and normalized FI divided by FI at 16 min in each villus ( right ) after initial loading of BODIPY - FA were quantified . For normalization of FI , villi that showed peak intensity earlier or later than 16 min were excluded ( n = 5 - 7 mice / group , 5 - 10 villi / mouse ) . Scale bars , 100 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.


Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Jejunum of vehicle - and ABX - treated mice were harvested 2 hours after oral lipid loading . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . CM , chylomicron ; IS , interstitium of lamina propria ; LC , large caveola ; Lu , lacteal lumen ; Ve , vesicle containing lipoproteins . Scale bars , 1 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.


quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice.


Comparisons of serum triglyceride ( TG ) and free fatty acids ( FFA ) at indicated time points in vehicle - and ABX - treated mice ( n = 5 - 7 mice / group ) .

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Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).


Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in right ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of 8 - week old SPF , GF or CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( C ) and VEGFR3 expression ( D , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from SPF , GF and CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum among SPF , GF and CONV mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Images and comparison of VEGFR3 expression ( presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum between WT and VR3iΔLEC mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and VR3iΔLEC mice . Note that , in addition to the alterations in the lacteal lengths in VR3iΔLEC mice , diameters of the lacteal also shrank by VEGFR3 ablation . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from WT and VR3iΔLEC mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum from WT and VR3iΔLEC mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Comparisons of serum triglyceride ( TG ) at indicated time points in WT and VR3iΔLEC mice ( n = 6 mice / group ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).


Comparison of protein levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in sorted CD45 - stromal cells and CD45 + MHCII + F4 / 80 + macrophages from jejunum and ileum of vehicle - treated mice . The mean of transcription levels of VEGF - C in CD45 - stromal cells was normalized to 1 , and the relative levels in macrophages were presented as fold change ( n = 4 mice / group ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).


Comparisons of VEGF - C transcription level in CD45 - stromal cells ( left ) and CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum and ileum of vehicle - and ABX - treated mice . The mean of transcription levels of VEGF - C in vehicle - treated mice was normalized to 1 , and the relative levels in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).


Images and comparison of F4 / 80 + macrophages in the villi of duodenum ( DD ) , jejunum ( JJ ) , and ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells ( E ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + cells ( F ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and DTR mice ( n = 5 mice / group ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from WT and DTR mice ( n = 5 mice / group ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and DTR mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and MyD88ΔMP mice ( n = 5 mice / group ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).


Comparisons of mRNA levels of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum and ileum of WT and MyD88ΔMP mice . The mean of transcription levels of VEGF - C in WT mice was normalized to 1 , and the relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and MyD88ΔMP mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A and rad59 ∆ rad52 - R70A cells . DNA was digested with Bsp1286I and probed with a MAT - distal sequence ( yellow box in panel B ) .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B).


Viability of indicated strains ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , and rad52 - R70A . DNA was digested with XhoI and EcoRI and probed with a Z sequence ( yellow box ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).


Percentage of chromosome loss of indicated mutants . Data information : χ2 test is used to determine the p - value and number of colonies tested per mutant is indicated in Appendix Table S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1.


Schematic of mutation analysis during allelic recombination ( left ) . Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site . Mutation rate ( right ) of WT , rad59 ∆ , rad52 - R70A was calculated as previously described ( Saini et . al . , 2013 ) . Data information : χ Mutation rates are reported as the median value , and statistical comparisons between median mutation rates were performed using the Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013). Data information: χ Mutation rates are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.


Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching ( H - 1400 assay ) .

O O O O O O O O O O O O O O O O O O O O O O

Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching (H-1400 assay).


Viability of indicated mutants are shown . ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Viability of indicated mutants are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


( A - C ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of WT and sir2 ∆ in the H - 150 assay ( A ) , H - - 150 no - gap assay ( B ) or native mating type switching H - 1400 assay ( C ) . ( mean ± SD ; n = 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A - C .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.


( A - B ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of indicated mutants in the H - 150 no - gap assay ( A ) or H - 150 assay ( B ) . ( mean ± SD ; n ≥ 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A and B .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of indicated mutants in the H-150 no-gap assay (A) or H-150 assay (B). (mean ± SD; n ≥3). Welch's unpaired t-test was used to determine the p-value in panels A and B.


HCT116 and U87 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

HCT116 and U87 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


HCT116 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

HCT116 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin ( Sim ) for 48h . Thereafter , lysates were collected and analyzed for total cholesterol levels . Shown are means and SD ( n = 3 ) .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin (Sim) for 48h. Thereafter, lysates were collected and analyzed for total cholesterol levels. Shown are means and SD (n = 3).


Shown is a volcano plot of patients from the TCGA ( database ) with high vs . low levels of ABCA1 . A close - up of the most significantly down - regulated genes is presented ( mRNAs related to the electron transport chain and mitochondrial metabolism ) . The x - axis highlights the fold changes ( log2 scale ) ( down - regulated genes , negative values ) , whereas the y - axis indicates the level of statistical significance ( shown in - log10 format ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Shown is a volcano plot of patients from the TCGA (database) with high vs. low levels of ABCA1. A close-up of the most significantly down-regulated genes is presented (mRNAs related to the electron transport chain and mitochondrial metabolism). The x-axis highlights the fold changes (log2 scale) (down-regulated genes, negative values), whereas the y-axis indicates the level of statistical significance (shown in -log10 format).


GBM12 GBM cells ( short term patient - derived xenograft ) , U87 GBM cells , HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263 , LXR623 or the combination for 72h . Thereafter , cellular viability was analyzed and statistical analysis was performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

GBM12 GBM cells (short term patient-derived xenograft), U87 GBM cells, HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263, LXR623 or the combination for 72h. Thereafter, cellular viability was analyzed and statistical analysis was performed.


U87 , T98G and LN229 cells were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 72h . Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263 , 10 µM LXR623 or the combination of both for 72h ( MeWo ) or for 48h ( HCT116 ) . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, Stem - like GBM cells , NCH644 , NCH421k and NCH690 were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 48h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

, Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


U87 ( 72h treatment ) and HCT116 ( 48h treatment ) were treated with 1 µM ABT263 , 20 µM ( U87 ) or 10 µM ( HCT116 ) LXR623 or the combination of both , stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential.


HCT116 colonic carcinoma cells were treated with ABT263 , LXR623 or the combination of both . Whole protein lysates were collected and subjected to capillary electrophoresis for the expression / cleavage of PARP , total caspase - 9 ( CP9 ) and Vinculin . CF : cleaved fragment , FL : full length .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.


HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3 - mimetics , WEHI - 539 ( Bcl - xL inhibitor ) , ABT199 ( Bcl - 2 inhibitor ) or A1210477 ( Mcl - 1 inhibitor ) in the presence or absence of LXR623 for 48h . Thereafter , cells were labeled with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h . Thereafter , protein lysates were collected and subjected to standard western blot ( LN229 , T98G and U87 ) or capillary electrophoresis ( U87 - EGFR - vIII and HCT116 ) for the expression of Usp9X , Mcl - 1 , Bcl - 2 , Bcl - xL , Noxa , BIM and / or Actin and Vinculin . In U87 - EGFRvIII and HCT116 , Vinculin was used in lieu of Actin .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.


LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with LXR623 as described in A . Thereafter , protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.


LN229 were transfected with siNT , siNoxa 1 , siNoxa 2 or BAK - siRNA for 72h . Thereafter , cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h . After conclusion of the treatment , cells were harvested , fixed , stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.


LN229 were transfected as described in B . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa , BAK and Vinculin .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O

LN229 were transfected as described in B. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin.


LN229 GBM cells were treated with 10 µM LXR623 ( LXR ) , 1 µM ABT263 or the combination for 48h . Thereafter , protein lysates were prepared and immunoprecipitated with an antibody against BAK . Standard western blotting was performed ( immunoprecipitation and the corresponding inputs ) with antibody against BAK and Mcl - 1 . The arrows highlight the specific protein bands , while stars indicate the immunoglobulin light - chains .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.


U87 cells were treated with 20 µM LXR623 for 7h and 24h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry . The lower left quadrant indicates the percentage of viable cells .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.


U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay . To account for dead cells , assays were normalized to DNA - content ( cell number ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number).


U87 cells were treated with LXR623 as indicated for 24h . Cell lysates were prepared and subjected to LC / MS for the determination of ATP levels .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels.


U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O

U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.


U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O

U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin ( G ) and by the standard western blot for the expression of Noxa and Actin .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin.


U87 cells were treated with increasing concentrations of LXR623 . Thereafter , whole cell protein lysates were prepared and analyzed for classical ER - stress markers , GRP78 , ATF4 and Noxa by capillary electrophoresis .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O

U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.


U87 cells were transfected with non - targeting siRNA ( n . t . - siRNA ) , ATF3 - siRNA , ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs . After 48h , cells were treated with LXR623 , whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3 , ATF4 , Noxa and Vinculin .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O

U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.


U87 cells were treated as in A . Shown are the levels of TCA - cycle metabolites .

U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non - labeled isotopologues ( m + 0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic acid and aspartic acid ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid and aspartic acid).


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of glutathione and nucleotides .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of glutathione and nucleotides.


U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool).


U87 cells were treated with DMSO or LXR623 20 µM for 24h . Thereafter , cells were processed for polar metabolite analysis by LC / MS . Shown are the levels of NAD and NADH2 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O

U87 cells were treated with DMSO or LXR623 20 µM for 24h. Thereafter, cells were processed for polar metabolite analysis by LC/MS. Shown are the levels of NAD and NADH2.


U87 cells were treated with LXR623 for 1h , 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( A - C ) . From this assay , coupled respiration was calculated ( D - F ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F).


U87 cells were treated with LXR623 , cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( G ) . From this assay , mitochondrial OCR and coupled respiration was calculated ( H - I ) .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I).