( D ) association of ubiquitinated 3HA - Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation . of indicated genotype expressing myc - tagged ubiquitin were grown as in ( C ) . Lysates were subjected to centrifugation on an optiprep density gradient . Collected fractions were analyzed by western blotting before after immunoprecipitation with anti - HA antibodies . Dpm1 Pgk1 were used as markers for bound soluble fractions respectively . Input fraction corresponds to 10 % of total amount used for IP . " Top " , " mid " , " bottom " indicate fractions from gradient . Asterisk marks a faster migrating unspecific band in input bottom fraction .

O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Membrane association of ubiquitinated 3HA-Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation. Cells of the indicated genotype and expressing myc-tagged ubiquitin were grown as in (C). Lysates were subjected to centrifugation on an optiprep density gradient. Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti-HA antibodies. Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively. Input fraction corresponds to 10% of the total amount used for IP. "Top", "mid", "bottom" indicate the fractions from the gradient. Asterisk marks a faster migrating unspecific band in the input bottom fraction.


( A ) Localization of chromosomally expressed GFP - Pgc1 from constitutive ADH1 promoter in wt doa10Δ . Arrowheads indicate GFP - Pgc1 labeling at ER , which is stained by Sec63 - Cherry . LDs were visualized upon with neutral lipid dye MDH . On bottom , box plot with quantitation of GFP - Pgc1 at envelope in wt doa10Δ . GFP - Pgc1 measurements were performed as described in Materials Methods section . Scale bar : 5 µm .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Localization of chromosomally expressed GFP-Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells. Arrowheads indicate GFP-Pgc1 labeling at the ER, which is stained by Sec63-Cherry. LDs were visualized upon staining with the neutral lipid dye MDH. On the bottom, box plot with quantitation of GFP-Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ. GFP-Pgc1 measurements were performed as described in the Materials and Methods section. Scale bar: 5 µm.


( C ) Crude membranes from wt doa10Δ expressing 3HA - Pgc1 were subjected to indicated treatments subsequently fractionated into pellet ( P ) supernatant ( S ) . ( D ) Crude membranes from wt doa10Δ expressing 3HA - GFP - Pgc1275 - 321 were analyzed as in ( C ) .

O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(C) Crude membranes from wt and doa10Δ cells expressing 3HA-Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet (P) and supernatant (S).(D) Crude membranes from wt and doa10Δ cells expressing 3HA-GFP-Pgc1275-321 were analyzed as in (C).


( A ) Localization of GFP - Pgc1 in are1Δ are2Δ lro1Δ dga1Δ in presence ( no LDs ) or absence of DOA10 ( no LDs doa10Δ ) . ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(A) Localization of GFP-Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence (no LDs) or absence of DOA10 (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( B ) GFP - Pgc1 was expressed under ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δ . microscopy was used to follow GFP - Pgc1 localization over time . LDs were visualized upon with neutral lipid dye MDH . Scale bar : 5 µm .

O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O

(B) GFP-Pgc1 was expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δ. Fluorescence microscopy was used to follow GFP-Pgc1 localization over time. LDs were visualized upon staining with the neutral lipid dye MDH. Scale bar: 5 µm.


( C ) Photo - conversion of tdEOS - Pgc1 expressed under ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ . red square marks photo - converted region . time ( in minutes ) after photoconversion is indicated . Arrowheads point LDs containing photoconverted tdEOS - Pgc1 . Scale bar 5µm .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O

(C) Photo-conversion of tdEOS-Pgc1 expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells. The red square marks the photo-converted region. The time (in minutes) after photoconversion is indicated. Arrowheads point LDs containing photoconverted tdEOS-Pgc1. Scale bar 5µm.


( D ) FRAP experiment of GFP - Pgc1 in wt doa10Δcells . Representative examples are shown . bleached areas are marked by red squares include a LD adjacent to ER . time ( in seconds ) after photobleaching is indicated . Each graph shows average intensities for 10 normalized to pre - bleached plotted over time . Error bars indicate standard deviation .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) FRAP experiment of GFP-Pgc1 in wt and doa10Δcells. Representative examples are shown. The bleached areas are marked by red squares and include a LD adjacent to ER. The time (in seconds) after photobleaching is indicated. Each graph shows average fluorescence intensities for 10 cells normalized to pre-bleached plotted over time. Error bars indicate standard deviation.


( A ) degradation of 3HA - Pgc1 was analyzed in with indicated genotype as in Fig . 1A . graph shows average of three independent experiments ; error bars represent standard deviation .

O O O O O O O B-GENEPROD O O 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) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) degradation of Doa10 substrates 3HA - Pgc1 3HA - Vma12 - Ndc10C ' was analyzed in of indicated genotypes treated with oleic acid . 3HA - Pgc1 3HA - Vma12 - Ndc10C ' were detected with anti - HA antibodies . Dpm1 was used as a loading control detected with anti - Dpm1 antibodies . graph shows average of three independent experiments ; error bars represent standard deviation .

O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( A ) Localization of GFP - tagged Pgc1 derivatives in which hydrophobic hairpin ( aa275 - 321 ) was replaced by anchor of Scs2 ( Pgc1Scs2MA ) or Bos1 ( Pgc1Bos1MA ) . ER LDs were visualized by expression of Sec63 - Cherry MDH , respectively . Scale bar : 5 µm .

O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O

(A) Localization of GFP-tagged Pgc1 derivatives in which the hydrophobic hairpin (aa275-321) was replaced by membrane anchor of Scs2 (Pgc1Scs2MA) or Bos1 (Pgc1Bos1MA). ER and LDs were visualized by expression of Sec63-Cherry and MDH staining, respectively. Scale bar: 5 µm.


( B ) degradation of 3HA - Pgc1 or indicated chimeras in wt doa10Δ was analyzed as in Fig . 1A . blot is representative of at least three independent experiments .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(B) The degradation of 3HA-Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig. 1A. The blot is representative of at least three independent experiments.


( C ) degradation of 3HA - GFP - Pgc1275 - 321 in wt doa10Δ was analyzed as in Fig . 1A . graph shows average of four independent experiments ; error bars represent standard deviation .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(C) The degradation of 3HA-GFP-Pgc1275-321 in wt and doa10Δ cells was analyzed as in Fig. 1A. The graph shows the average of four independent experiments; error bars represent the standard deviation.


( D ) Localization of 3HA - GFP - Pgc1275 - 321 in doa10Δ . ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(D) Localization of 3HA-GFP-Pgc1275-321 in doa10Δ cells. The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( A ) degradation of 3HA - Pgc1 - GPAT160 - 216 , containing GPAT4 hydrophobic hairpin ( aa160 - 216 ) replacing one of Pgc1 was analyzed in with indicated genotype as in Fig . 1A . graph shows average of three independent experiments ; error bars represent standard deviation .

O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O 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) The degradation of 3HA-Pgc1-GPAT160-216, containing GPAT4 hydrophobic hairpin (aa160-216) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) Localization of GFP - Pgc1 - GPAT4160 - 216 in with indicated genotype . Arrowheads indicate GFP - Pgc1 - GPAT4160 - 216 labeling at ER , marked with Sec63 - Cherry . LDs were visualized upon with MDH . Scale bar : 5 µm .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O

(B) Localization of GFP-Pgc1-GPAT4160-216 in cells with the indicated genotype. Arrowheads indicate GFP-Pgc1-GPAT4160-216 labeling at the ER, marked with Sec63-Cherry. LDs were visualized upon staining with MDH. Scale bar: 5 µm.


( C ) degradation of 3HA - Pgc1 - GPAT160 - 216 in with indicated genotype was analyzed as in Fig . 1A . graph shows average of three independent experiments ; error bars represent standard deviation .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The degradation of 3HA-Pgc1-GPAT160-216 in cells with the indicated genotype was analyzed as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( D ) Localization of GFP - Pgc1 - GPAT160 - 216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant ( no LDs doa10Δ ) . ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(D) Localization of GFP-Pgc1-GPAT160-216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


B Representative microphotographs quantitative analyses of ACE2 immunohistochemical in lungs from young ( 2 months ) old ( 22 months ) mice ( n = 3 mice per group ) . Scale bar 200 µm .

O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O

B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.


C Representative microphotographs quantitative analyses of ACE2 immunohistochemical in lung parenchyma of young ( 20 - 35 years old ) old ( 60 - 80 years old ) humans ( n = 4 - 7 individuals per group ) . Scale bar , 200 µm .

O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.


D Double - marker immunofluorescence quantitative analyses of ACE2 level in type II pneumocytic pro - SP - C - positive CD31 - positive endothelia in lungs from young old mice ( n = 3 - 6 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


E Double - marker immunofluorescence quantitative analyses of ACE2 level in type II pneumocytic TTF1 - positive CD31 - positive endothelia in lungs from young old humans ( n = 4 - 7 individuals per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.


C Representative quantitative analyses of ACE2 immunohistochemical in lungs of - matched wild type G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 200 µm .

O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O

C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.


D Double - marker immunofluorescence quantitative analyses of ACE2 level in type II pneumocytic pro - SP - C - positive CD31 - positive endothelia in lungs of - matched wild type G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F / F at indicated time points following 4OHT treatment consequent TRF2 knockout . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O O O

A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 at indicated time points following doxycycline treatment consequent TRF2 knockdown . Scale bar , 25 µm . D RT - qPCR detection of ACE2 mRNA expression in HeLa shTRF2 treated as in C ( n = 6 independent experiments ) .

O B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O

C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).


E Representative immunofluorescence of 53BP1 in liver from Trf2F / F mice treated with tamoxifen ( to induce TRF2 loss telomere uncapping ) or vehicle . as been injected also with PBS . Scale bar , 10 µm . F RT - qPCR detection of Ace2 mRNA expression in livers of mice treated as in E ( n = 5 - 7 mice per group ) .

O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O

E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).


B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O 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 Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.


A Immunofluorescence showing gammaH2AX foci in Trf2F / F MEFs at indicated time points following 4OHT treatment consequent TRF2 knockout treated with DMSO or ATMi . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O O O

A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Representative immunofluorescence of 53BP1 in liver from Trf2F / F mice treated with tamoxifen ( to induce telomere uncapping ) or vehicle injected with indicated ASOs or PBS as control . Scale bar , 10 µm . D RT - qPCR detection of Ace2 mRNA in livers of mice treated as in C ( n = 5 - 8 mice per group ) .

O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O

C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).


E Representative microphotographs quantitative analyses of ACE2 immunohistochemical in lungs of - matched wild type G3 Terc - / - mice , treated with indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 200 µm .

O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O

E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.


F Double - marker immunofluorescence quantitative analyses of ACE2 level in type II pneumocytic pro - SP - C - positive in lungs of - matched wild type G3 Terc - / - mice , treated with indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


( A ) Profile of transcript of autophagy - related genes in iBAT which are significantly modified in cold exposed mice ( 4ºC , 24h ) , compared with mice maintained at thermoneutral ( 29ºC ) . ( Fisher ' s exact corrected by Benjamini - Hochberg method , p < 0 . 05 ) . represents log - fold change of RPKM values of cold versus thermoneutral ( TN ) sample data obtained from RNAseq . Each square represents replicates ( n = 4 ) . most repressed genes are shown on bottom of .

O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(A)Profile of transcript levels of autophagy-related genes in iBAT which are significantly modified in cold exposed mice (4ºC, 24h), compared with mice maintained at thermoneutral temperature (29ºC). (Fisher's exact test corrected by the Benjamini-Hochberg method, p< 0.05). Heatmap represents log-fold change of RPKM values of cold versus thermoneutral (TN) sample data obtained from RNAseq analysis. Each square represents replicates (n=4). The most repressed genes are shown on the bottom of the heatmap.


Parkin expression in iBAT from mice exposed to cold ( 4ºC ) for 1 day ( 1d ) or 21 days ( 21d ) compared with control mice ( ctl ) maintained at 22ºC . Left : Relative transcript . Right : representative immunoblot Parkin . Ponceau ( PS ) was used as loading control . ( n = 6 ; transcript ) ( n = 3 ; ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control # # p < 0 . 01 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc .

B-GENEPROD B-EXP_ASSAY O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Parkin expression in iBAT from mice exposed to cold (4ºC) for 1 day (1d) or 21 days (21d) compared with control mice (ctl) maintained at 22ºC. Left: Relative transcript levels. Right: representative immunoblot and Parkin protein quantification. Ponceau staining (PS) was used as loading control. (n=6; transcript levels) (n=3; protein levels). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ##p<0.01, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript of Park2 in sWAT eWAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control # # # p < 0 . 001 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc .

O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 in sWAT and eWAT. (n= 6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ###p<0.001, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript of Parkin in iBAT , sWAT eWAT of mice injected with β3 - AR agonist CL316 , 243 , every day for 1 week . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * * p < 0 . 001 , CL316 , 243 treatment vs . saline . Two - unpaired Student ' s t - .

O O O B-GENEPROD O B-TISSUE O B-TISSUE I-TISSUE O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O

Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 Ucp1 in mature brown adipocytes compared with stromal vascular fraction ( SVF ) in iBAT . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - unpaired Student ' s t - .

O O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction (SVF) in iBAT. (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


mRNA expression profiles of Park2 Ucp1 during differentiation . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to day ( d ) 4 . Two - unpaired Student ' s t - .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression profiles of Park2 and Ucp1 during differentiation. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to day (d) 4. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 Ucp1 in brown adipocytes in culture with bovine serum ( FBS , to trigger in vitro differentiation ) or charcoal - stripped serum ( CSS , non - permissive for differentiation ) . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - unpaired Student ' s t - .

O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum (FBS, to trigger in vitro differentiation) or charcoal-stripped serum (CSS, non-permissive for differentiation). (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 Ucp1 in brown adipocytes in culture treated with norepinephrine ( NE ) or cAMP for indicated times . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to non - treated . One - way ANOVA with Dunnett ' s post hoc .

O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine (NE) or cAMP for indicated times. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Parkin in brown adipocytes treated with NE or cAMP for indicated times in presence or absence of 3 - methyladenine ( 3 - MA ) . Top : Representative immunoblots . p62 LC3BII are shown as positive controls of 3 - MA treatment . β - actin was use as loading control . Bottom : Parkin ( performed using 3 immunoblots processed in parallel due to sample ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to NE / cAMP non - treated . One - way ANOVA with Dunnett ' s post hoc .

B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3-methyladenine (3-MA). Top: Representative immunoblots. p62 and LC3BII proteins are shown as positive controls of 3-MA treatment. β-actin was use as loading control. Bottom: Parkin protein quantification (performed using 3 immunoblots processed in parallel due to sample size). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to NE/cAMP non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Relative transcript of Park2 in brown adipocytes treated with NE in presence or absence of cycloheximide ( CHX ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Two - unpaired Student ' s t - .

O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide (CHX). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative mRNA expression of Parkin in brown adipocytes treated with NE in presence or absence of actinomycin D ( n = 3 ) . Data are presented as means ± s . e . m . One - way ANOVA with Tukey ' s post hoc comparing treatments .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D (n=3). Data are presented as means ±s.e.m. One-way ANOVA with Tukey's post hoc test comparing treatments.


Park2 gene promoter activity in HIB - 1B transfected with a pGL4 - Park2 - Luc reporter construct treated with or without NE or cAMP . Data was normalized using Renilla luciferase activity are expressed as relative firefly luciferase activity compared to that in untreated . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated . ANOVA with Dunnett ' s post hoc

B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct treated with or without NE or cAMP. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells. (n=6). Data are presented as means ±s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test


Relative transcript of Park2 Ucp1 in brown adipocytes treated with cAMP ( 12 h ) plus H89 ( a PKA inhibitor ) or SB202190 ( a p38 MAPK inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 with vs . without inhibitor # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - unpaired Student ' s t - .

O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP (12 h) plus H89 (a PKA inhibitor) or SB202190 (a p38 MAPK inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 , of glycerol in culture medium of brown adipocytes treated with cAMP ( 24h ) plus Atglistatin ( an adipose triglyceride lipase inhibitor ) or CAY10499 ( a hormone sensitive lipase inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 with vs . without inhibitor # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - unpaired Student ' s t - .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O

Relative transcript levels of Park2, and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP (24h) plus Atglistatin (an adipose triglyceride lipase inhibitor) or CAY10499 (a hormone sensitive lipase inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 Ucp1 in brown adipocytes treated for 24h with a PPARα activator ( GW7647 ) , a PPARα inhibitor ( GW6471 ) with or without norepinephrine ( NE ) , or - trans - retinoic acid ( RA ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 . Two - unpaired Student ' s t - .

O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator (GW7647), a PPARα inhibitor (GW6471) with or without norepinephrine (NE), or all-trans-retinoic acid (RA). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 in wild - type PPARα - KO brown adipocytes treated for 24h with NE ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc .

O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 in wild-type and PPARα-KO brown adipocytes treated for 24h with NE (n=3). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Park2 gene promoter activity in HIB - 1B transfected with a pGL4 - Park2 - Luc reporter construct , when indicated , with a pSG5 - Ppara expression vector . were treated with PPARα activator GW7647 or NE . Data was normalized using Renilla luciferase activity are expressed as relative firefly luciferase activity compared to that only transfected with pGL4 - Park2 - Luc construct ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated . ANOVA with Dunnett ' s post hoc .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct and, when indicated, with a pSG5-Ppara expression vector. Cells were treated with the PPARα activator GW7647 or NE. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4-Park2-Luc construct (n=6). Data are presented as means ± s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test.


Left : Representative optical microscopic of H & E - stained iBAT ( scale bars , 110µm ) electron microscopic of iBAT ( scale bars , 20µm ) from wild - type ( WT ) Parkin - KO ( KO ) mice . Right : of lipid droplet from electron microscopic ( 10 per condition were used , n = 3 WT , n = 3 Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . Two - unpaired Student ' s t - .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Representative optical microscopic images of H&E-stained iBAT (scale bars, 110µm) and electron microscopic images of iBAT (scale bars, 20µm) from wild-type (WT) and Parkin-KO (KO) mice. Right: Quantification of lipid droplet area from electron microscopic images (10 images per condition were used, n =3 WT, n=3 Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. Two-tailed unpaired Student's t-test.


Representative thermal ( left ) of surface ( right ) for interscapular region . ( n = 6 ) . Data are presented as means ± s . e . m . * * * p < 0 . 001 . Two - unpaired Student ' s t - .

O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O 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 thermal images (left) and quantification of surface temperature (right) for the interscapular region. (n=6). Data are presented as means ±s.e.m. ***p<0.001. Two-tailed unpaired Student's t-test.


Representative immunoblot of UCP1 loading control ( Ponceau , PS ) in iBAT samples .

O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O

Representative immunoblot of UCP1 protein and the loading control (Ponceau staining, PS) in iBAT samples.


Relative transcript of thermogenesis - related genes of iBAT from WT Parkin - KO mice exposed to acute cold ( 4ºC , 24h ) compared to those maintained at control ( 22ºC ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , CTL vs . cold ; # p < 0 . 05 , WT vs . Parkin - KO . ANOVA with Tukey ' s post hoc .

O O O O O O O O B-TISSUE O O B-GENEPROD O O B-ORGANISM O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O

Relative transcript levels of thermogenesis-related genes of iBAT from WT and Parkin-KO mice exposed to acute cold (4ºC, 24h) compared to those maintained at the control temperature (22ºC). (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001, CTL vs. cold; #p<0.05, WT vs. Parkin-KO. ANOVA with Tukey's post hoc test.


gain from WT Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

B-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM O O 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-GENEPROD O O O O

Weight gain from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


, blood glucose plasma insulin from WT Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

O B-TISSUE B-SMALL_MOLECULE B-TISSUE B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O 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-GENEPROD O O O O

, blood glucose levels and plasma insulin levels from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


insulin tolerance ( ITT ) from WT Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O 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-GENEPROD O O O O

insulin tolerance test (ITT) from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


Relative transcript of thermogenesis - related genes in iBAT from WT Parkin - KO mice given HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . # p < 0 . 05 . Two - unpaired Student ' s t - .

O O O O O O O O B-TISSUE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of thermogenesis-related genes in iBAT from WT and Parkin-KO mice given HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. #p<0.05. Two-tailed unpaired Student's t-test.


Relative transcript of Park2 in iBAT from WT mice fed CD or HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . Two - unpaired Student ' s t - .

O O O B-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. Two-tailed unpaired Student's t-test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Top : Total content in iBAT during cold deaclimation . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to 21d . ANOVA with Dunnett ' s post hoc . Bottom : Representative optical microscopic of H & E - stained iBAT ( scale bars , 100µm ) electron microscopic of iBAT ( scale bars , 10µm ) .

O B-ORGANISM O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Top: Total protein content in iBAT during cold deaclimation. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to 21d. ANOVA with Dunnett's post hoc test. Bottom: Representative optical microscopic images of H&E-stained iBAT (scale bars, 100µm) and electron microscopic images of iBAT (scale bars, 10µm).


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Relative transcript of thermogenesis - related genes Park2 in iBAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to 21d . ANOVA with Dunnett ' s post hoc .

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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Relative transcript levels of thermogenesis-related genes and Park2 in iBAT. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to 21d. ANOVA with Dunnett's post hoc test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of indicated thermogenic autophagic in iBAT during cold acclimation deacclimation . PS : Ponceau .

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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation. PS: Ponceau staining.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of Parkin phosphoSer65 total Parkin in whole lysates of iBAT after Parkin immunoprecipitation . Arrowheads indicate Parkin 52kDa band asterisk indicates a non - specific band .

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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation. Arrowheads indicate Parkin 52kDa band and asterisk indicates a non-specific band.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative electron microscopic of autophagic mitophagic events in iBAT after 1 day of cold deacclimation . Yellow arrowheads indicate mitochondrial cristae , blue arrowheads indicate double - autophagosomes , red arrowheads indicate structures compatible with mitochondria - derived vesicles . LD : lipid droplet . Scale bars , 1 µm .

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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation. Yellow arrowheads indicate mitochondrial cristae, blue arrowheads indicate double-membrane autophagosomes, and red arrowheads indicate structures compatible with mitochondria-derived vesicles. LD: lipid droplet. Scale bars, 1 µm.


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative immunoblot of LC3B in iBAT , loading control ( Ponceau , PS ) .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative immunoblot of LC3B in iBAT, and the loading control (Ponceau staining, PS).


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Immunoblot for p62 OPTN ( left ) its ( right ) in iBAT . PS was used as loading control . Arrowhead indicates p62 band asterisk indicates a non - specific band . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * * p < 0 . 01 . ANOVA with Tukey ' s post hoc .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Immunoblot for p62 and OPTN (left) and its quantification (right) in iBAT. PS was used as the loading control. Arrowhead indicates p62 band and asterisk indicates a non-specific band. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. **p<0.01. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . of total amounts of indicated OXPHOS subunits UCP1 in iBAT . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Citrate synthase activity in iBAT extracts . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Citrate synthase activity in iBAT protein extracts. (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Mitochondrial ( mtDNA ) content in iBAT relative to ( nDNA ) . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc .

O O O O O O B-GENEPROD O O O O O B-ORGANISM 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-SUBCELLULAR O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Mitochondrial DNA (mtDNA) content in iBAT relative to nuclear DNA (nDNA). (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative electron microscopic of mitochondrial integrity autophagic / mitophagic events in iBAT after 1 day of cold - deacclimation . Yellow arrowheads indicate mitochondria cristae blue arrowheads indicate double - autophagosomes . Left panels scale bars , 2 µm . Right panels scale bars , 1 µm .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative electron microscopic images of mitochondrial integrity and autophagic/mitophagic events in iBAT after 1 day of cold-deacclimation. Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double-membrane autophagosomes. Left panels scale bars, 2 µm. Right panels scale bars, 1 µm.


Sexing established ESCs by detecting Y - linked Zfy gene using genomic ( gDNA ) - qPCR , normalized to Gapdh . n . d . : not detected . Average of presumed was set to 1 .

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Sexing the established ESCs by detecting the Y-linked Zfy gene using genomic DNA (gDNA)-qPCR, normalized to Gapdh. n.d.: not detected. Average of presumed males was set to 1.


- FISH detection of number of X - linked Atrx gene foci . Representative of - FISH are shown . Nuclei are surrounded by dotted lines . Magenta : Atrx ( arrowheads ) , cyan : DAPI . N > 100 .

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DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.


from - seq . Each log10 ( FPKM + 1 ) value was used for clustering . Genes with high or low expression are shown in red blue , respectively . FPKM : fragments per kilobases per million reads .

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Heatmap analysis from RNA-seq. Each log10(FPKM+1) value was used for clustering. Genes with high or low expression are shown in red and blue, respectively. FPKM: fragments per kilobases per million reads.


X Y chromosome mapping . Genes with high expression ( up ) are shown in orange low expression ( down ) are shown in blue . defined of up or down genes is > 1 . 5 < two - thirds , respectively . comparison is ( each vs average for ) or ( each vs average for ) . location controls for expressed genes ( exp . genes ) on X - chromosome Y - chromosome are plotted ( red squares ) for genes whose FPKM averages were above 1 for or .

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X and Y chromosome mapping. Genes with high expression (up) are shown in orange and low expression (down) are shown in blue. The defined ratio of up or down genes is > 1.5 and < two-thirds, respectively. The comparison is (each female vs the average for males) or (each male vs the average for females). The location controls for the expressed genes (exp. genes) on the X-chromosome and Y-chromosome are plotted (red squares) for genes whose FPKM averages were above 1 for females or males.


Clonogenic for ESCs in response to etoposide ( A ) or camptothecin ( B ) . Non - treated were set to 1 .

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Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.


Nanog HR . of GFP - positive colonies after gene targeting to Nanog locus is shown . results for each are shown at right .

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Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown. The results for each cell line are shown at right.


Nanog expression confirmed by RNA-seq.


Detection of number of X - linked Atrx gene foci by - FISH in GFP - positive subclones of Nanog - targeting ( nine independent clones , N > 100 each ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH in GFP-positive subclones of the Nanog-targeting assay (nine independent clones, N> 100 each), performed as described in Figure 1C.


Sexing of established ESCs ( gDNA - qPCR , Zfy / Gapdh ) , performed as described in Figure 1B .

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Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B.


Detection of number of X - linked Atrx gene foci by - FISH with or without dox addition for 96 h ( N > 100 ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.


Detection of Xist cloud in # B3 by - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image is shown [ magenta : Xist ( arrowhead ) , cyan : DAPI . Scale bar : 10 μm ] .

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Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].


Allele - distinguishable RT - PCR for detecting Hprt , G6pdx , Rnf12 X - linked genes . Bands with anticipated sizes expressed from XJF1 or XTX alleles are shown .

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Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown.


Nanog HR . of GFP - positive colonies after gene targeting to Nanog locus is shown as described in Figure 2C . # B2 or # B3 were cultured with or without dox addition for 96 h . combined number of GFP - positive total colonies from experiments are listed summarized at right .

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Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.


Detection of Xist foci by - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image with of Xist - positive is shown [ magenta : Xist , cyan : DAPI . ] .

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Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].


Detection of number of X - linked Atrx gene foci by - FISH , with or without dox addition for 96 h ( N > 100 each ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.


Estimated repair events . flow cytometry plots are shown in Figure EV4 . of GFP - positive in TX - DRGFP # 11 TX - EJ5GFP # 23 was normalized to transfection efficiency detected by VENUS expression vector . Dox ( - ) was set to 1 of relative GFP - positive is shown ( n = 3 ) .

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Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).


Volcano plot of DEGs in ESCs ( # A4 , # A10 , # A11 ) versus ( # A6 , # A13 , # A17 ) . definition of up ( red dots ) or down ( blue dots ) genes is as follows : of up or down genes is > 1 . 5 < two - thirds , respectively , with q - value < 0 . 05 .

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Volcano plot of DEGs in female ESCs (#A4, #A10, #A11) versus males (#A6, #A13, #A17). The definition of up (red dots) or down (blue dots) genes is as follows: the ratio of up or down genes is > 1.5 and < two-thirds, respectively, with q-value< 0.05.


Brcc3 expression confirmed by - seq [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

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Brcc3 expression confirmed by RNA-seq [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by RT - qPCR [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

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Brcc3 expression confirmed by RT-qPCR [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by western blotting [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

Brcc3 expression confirmed by western blotting [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Confirmation of Brcc3 expression from knockdown samples in by RT - qPCR ( technical duplicates )

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Confirmation of Brcc3 expression from knockdown samples in by RT-qPCR (technical duplicates)


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Confirmation of Brcc3 expression from knockdown samples in western blotting


Nanog HR with Brcc3 knockdown ( three biological replicates ) . of GFP - positive colonies after gene targeting to Nanog locus is shown , as shown in Figure 2C . combined number of GFP - positive total number of colonies are shown ( please note that total number of # A13 for drug screening was 3 . 67 × 107 because of contamination of 1 out of 12 dishes , compared with other colonies for which 4 × 107 were used ) .

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Nanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).


Kaplan - Meier curves for overall survival of breast cancer on BRCC3 ( A ) , XIST ( B ) , BRCC3 / XIST ( C ) expression . HR : hazard .

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Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio.


for collagen inserts in panel B represent hematoxylin ( nucleus ) & eosin ( cytoplasm ) for same tissue of collagen In cases n = 4 control mice , ≥ 3 mice for 2mg 5 mg ) for each is shown in histogram in right

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IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right side


fo aSMA , CD68 , CD204 For aSMA , CD68 , CD204 of positive is relative to control condition . In cases n = 4 control mice , ≥ 3 mice for 2mg 5 mg ) Scale bar : 100 mm . for each is shown in histogram in right

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IHC images fo aSMA, CD68, and CD204 For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side


Representative traces tracking bead displacements Histogram shows relative bead displacement for first last , n = 27 control n = 37

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Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifen


Histogram shows of gel contraction , n > 10 gels per

Histogram shows percentage of gel contraction, n>10 gels per condition


Representative mechanosensing trace of PSC treated with tamoxifen Estrogen receptor ( ER antagonists Histogram shows relative bead displacement for first last , n > 20 for

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Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


Representative mechanosensing trace of PSC treated with tamoxifen an G coupled - estrogen receptor ( GPER ) antagonists Histogram shows relative bead displacement for first last , n > 20 for

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Representative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


Histogram shows of gel contraction by PSCs treated with tamoxifen in presence of indicated

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Histogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonist


Histogram shows of gel contraction after treatment with indicated agonist , n > 10 gels per

Histogram shows percentage of gel contraction after treatment with indicated agonist, n>10 gels per conditio


Young " s modulus for matrices previously remodeled by PSCs ; control n = 126 tamoxifen n = 145 . Lines error bars indicate mean ± s . e . m . Four experimental

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Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental repl


Representative of H & E showing AsPC1 cancer invasions in matrices previously remodeled by control PSCs ( top ) or tamoxifen treated PSCs ( bottom ) , scale bar 50 μm AsPC1 invasion , central box represents values from lower to upper quartile . middle represents mean . vertical extends from minimum to maximum value , n = 30 control n = 40

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Representative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen


Immunofluorescence images of PSCs stained for YAP


of / cytoplasm YAP in PSCs ( four experimental replicates )

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Quantification of the nuclear/cytoplasm YAP in PSCs (four experimental replicates)


qPCR mRNA for YAP target genes connective tissue grow factor ( CTGF ) Ankyrin Repeat Domain 1 ( ANRKD1 ) , ( three experimental replicates )

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qPCR mRNA levels for YAP target genes connective tissue grow factor (CTGF) and Ankyrin Repeat Domain 1 (ANRKD1), (three experimental replicates)


Western blot bands for YAP , pS127 YAP total . ( E ) of YAP pYAP Ser127 normalised to total , expressed relative to unstimulated control ( n = 8 )

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Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8)


for YAP in KPC mice pancreatic tissues ( control treated with 2 5 mg tamoxifen ) , YAP is brown . Scale bar : 100

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IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μm


of YAP positive nucleus . n = 5 control mice , ≥ 3 mice for 2mg 5

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Quantification of YAP positive nucleus. n=5 control mice, and ≥3 mice for 2mg and 5 mg


IF for total ( MLC - 2 ) phosphorylated ( pMLC - 2 ) in PSCs , scale bar is 20

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IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μm


of in A . Each data point represents a Western blot for total phosphorylated mlc -

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Quantification of staining in A. Each data point represents a cell Western blot for total and phosphorylated mlc-2


of total active RhoA , expressed as of total RhoA in control

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Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control condition


for phosphorylated mlc - 2 in mice pancreatic tissues ( n = 5 control , n = 5 2mg , n = 3 5mg ) . Scale bar : 100 μm of in

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IHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm Quantification of staining in E


D Immunofluorescence of - damage - associated PARP . show adjacent normal crypts of tissue P009T , marked by N T , respectively , tissue of P008 P009 , as indicated . Scale bar 100µm . Significance was assessed by an unpaired t - , after blinded of 10 random per .

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D Immunofluorescence of DNA-damage-associated nuclear protein PARP. Images show adjacent normal and tumor crypts of tissue P009T, marked by N and T, respectively, and tumor tissue of patients P008 and P009, as indicated. Scale bar 100µm. Significance was assessed by an unpaired t-test, after blinded analysis of 10 random images per tumor.


E Activities of Wnt / β - catenin MAPK target genes in organoid single transcriptomes , ordered along latent time .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time.


F Activities of TFF3 , FABP1 , MKI67 MMP7 in organoid single transcriptomes , ordered along latent time .

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F Activities of TFF3, FABP1, MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time.


A Gene expression of LGR5 EPHB2 , along activity gradients of LGR5 - ISC or MAPK target gene signatures .

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A Gene expression of LGR5 and EPHB2, along activity gradients of LGR5-ISC or MAPK target gene signatures.


C of key CyTOF data . Average activities of selected analytes are given as log fold - change after normalization to DMSO control condition . Range of color scale was adjusted for each analyte . For relative changes between analytes , see Appendix Fig . S7 ) .

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C Heatmap of key CyTOF data. Average activities of selected analytes are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each analyte. For relative changes between all analytes, see Appendix Fig. S7).


F Scaled signature expression single gene expression moments per , under control ( grey ) preferred treatment ( color ) conditions , as indicated . 10 % of populating end of latent time are displayed in bold . It is of note that latent time is linear thus cannot capture multiple populations located at developmental end points . Densities at side lines display expression in .

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F Scaled signature expression and single gene expression moments per cell, under control (grey) and preferred treatment (color) conditions, as indicated. 10% of cells populating the end of latent time are displayed in bold. It is of note that latent time is linear and thus cannot capture multiple populations located at developmental end points. Densities at the side lines display expression in all cells.


( C ) Graph showing log10 CFU / at 4 days post - inoculation with indicated strains ( n ≥ 5 ) . Each circle represents values obtained in , whereas mean ± s . d . is represented with lines inside each group . No statistical differences were found between infection groups using a one - sided ANOVA followed by post - hoc Fisher ' s PLSD .

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(C) Graph showing log10 CFU/gland at 4 days post-inoculation with the indicated strains (n≥5). Each circle represents the values obtained in individual animals, whereas mean ± s.d. is represented with lines inside each group. No statistical differences were found between infection groups using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test.


Plots showing RT - qPCR conducted to quantify relative expression of indicated interleukins coding genes in mammary . Results are expressed as mean ± s . d ( n = 10 for PBS , n = 5 for WT n = 4 for CV2 ) of 2 - ΔΔCt relative expression values of indicated interleukins ; value from each was calculated from three technical replicates . values obtained in PBS group were used as control for normalization of gene expression ( = 1 ) . Statistical was performed using a one - sided ANOVA followed by post - hoc Fisher ' s PLSD : ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 005 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).


( B ) Comparison of turbidimetric results obtained with mpn142 mpn142Opt secretion signals . Culture supernatants ( n = 1 ) were collected at 0 h , 5 h or 24 h post - inoculation as indicated . WT medium ( Hayflick ) were added as controls .

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(B) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals. Culture supernatants (n=1) were collected at 0 h, 5 h or 24 h post-inoculation as indicated. WT strain and medium (Hayflick) were added as controls.


( A , B ) Mature S . aureus biofilms were allowed to develop for 24 h in polystyrene plates then treated for indicated time intervals with suspensions , or culture supernatants of CV2 or CV2 - DispB strains ( A ) or WT or WT - DispB ( B ) . Biofilm presence was assessed by crystal violet included a negative control ( i . e . , crystal violet without biofilm ) . results are expressed as mean ± s . d . of OD 595 nm absorbance values obtained from three different biological replicates ( n = 3 ) statistically compared by one - sided ANOVA followed by post - hoc Fisher ' s PLSD : * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 ; * * * * P ≤ 0 .

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(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005


( C , D ) Plots showing results obtained from in vitro or ex vivo dispersion assays with indicated strains from control . Each circle represents OD 595 nm values obtained for catheters ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Results from one - sided ANOVA followed by Fisher ' s PLSD are shown for treatments statistically different from those on strains not secreting dispersin B ( i . e . , WT or CV2 strains ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control. Each circle represents the OD 595 nm values obtained for individual catheters (n≥4), whereas mean ± s.d. is represented with lines inside each group. Results from one-sided ANOVA followed by Fisher's PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B (i.e., the WT or CV2 strains). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( C , D ) Plots showing SUV60 variation ( % ) between day 1 day 4 of different treatments . Each circle represents SUV60 variation obtained for ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Data below dotted lines indicate that SUV 60 values decreased at D4 post - treatment . Results from one - sided ANOVA followed by Fisher ' s PLSD are shown . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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(C, D) Plots showing the SUV60 variation (%) between day 1 and day 4 of the different treatments. Each circle represents the SUV60 variation obtained for individual animals (n≥4), whereas mean ± s.d. is represented with lines inside each group. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Results from one-sided ANOVA followed by Fisher's PLSD test are shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( G ) Baf A1 sensitivity for NDST3 KO after NDST3 restoration . NDST3 KO with stable expression of Flag - NDST3 or Flag - EGFP ( control ) were treated with 25 nM Baf A1 for 72 h , survival was measured using calcein AM . survival was calculated as of treated relative to untreated ( n = 4 independent cultures , * * * * P < 0 . 0001 ) . Data information : Error bars represent ± standard deviation . Scale bar , 200 μm .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration. The NDST3 KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001). Data information: Error bars represent ± standard deviation. Scale bar, 200 μm.


( C ) Lysosomal pH values in WT NDST3 KO RPE1 calculated from of LysoSensor Yellow / Blue dextran against pH calibration curve in ( B ) ( n = 3 independent cultures , * P = 0 . 0242 ) . ( D ) Lysosomal pH values measured for WT NDST3 KO RPE1 after treatment with Baf A1 ( 100 nM , 1h ) ( n = 3 independent cultures , * P = 0 . 0305 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

O O O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-SUBCELLULAR O O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( F ) Magic Red for Cat B proteolytic activity . WT NDST3 KO RPE1 were treated with vehicle ( VEH ) or Baf A1 ( 100 nM , 1 h ) then stained with MR - ( RR ) 2 to measure Cathepsin B activity ( n = 48 in VEH - treated WT group , n = 37 in VEH - treated NDST3 KO group , n = 64 in Baf A1 - treated WT group , n = 42 in Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

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(F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( G ) Alexa Fluor ( AF ) 488 - dextran degradation . WT NDST3 KO RPE1 were loaded with AF488 - dextran in culture medium observed for . AF488 signals without a chase period , with a 4 - h chase period , with 4 - h chase period in presence of Baf A1 ( 100 nM , 1h ) were recorded as of uptake , chase , chase with Baf A1 , respectively . AF488 per was quantified calculated into degradation according to equation in Materials Methods ( n = 62 in VEH - treated WT group , n = 36 in VEH - treated NDST3 KO group , n = 65 in Baf A1 - treated WT group , n = 78 in Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY 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-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O

(G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488 fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated NDST3 KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( J ) LC3 turnover after a 48 - h Baf A1 treatment . WT NDST3 KO RPE1 were cultured with 25 nM Baf A1 for 48 h analyzed for LC3B - II fold change as in ( I ) ( n = 6 independent experiments , * * P = 0 . 0014 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) The LC3 turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( A ) To examine of V - ATPase V1 - V0 holoenzyme in WT NDST3 KO RPE1 , fractions were analyzed by immunoblotting using antibodies against V - ATPase subunits V1A ( ATP6V1A ) , V1C1 ( ATP6V1C1 ) , V0D ( ATP6V0D ) . LAMP1 vinculin were used as loading controls for , respectively . ( B ) of of ATP6V0D relative to LAMP1 in fraction from ( A ) ( n = 3 independent experiments , P = 0 . 7786 ) . Data information : Error bars represent ± standard deviation .

O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O 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) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively. (B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786). Data information: Error bars represent ± standard deviation.


( A , B ) Perinuclear enrichment colocalization of acetylated microtubules , lysosomes , V - ATPase V1 subunits . RPE1 stably expressing LAMP1 - GFP were treated with vehicle ( VEH ) or 10 μg / mL nocodazole ( Noco ) for 6 h before being fixed subjected to immunofluorescence with antibodies against ATP6V1C1 acetylated - α - tubulin ( Ac - α - tubulin ) . nucleus was stained with DAPI . Representative of LAMP1 - GFP , ATP6V1C1 , Ac - α - tubulin immunostaining are shown in ( A ) . Representative distribution profiles of Ac - α - tubulin , LAMP1 - GFP ATP6V1C1 were plotted by Fiji software in ( B ) .

O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O

(A, B) Perinuclear enrichment and colocalization of acetylated microtubules, lysosomes, and V-ATPase V1 subunits. RPE1 cells stably expressing LAMP1-GFP were treated with vehicle (VEH) or 10 μg/mL nocodazole (Noco) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated-α-tubulin (Ac-α-tubulin). The nucleus was stained with DAPI. Representative images of LAMP1-GFP, ATP6V1C1, and Ac-α-tubulin immunostaining are shown in (A). Representative distribution profiles of Ac-α-tubulin, LAMP1-GFP and ATP6V1C1 were plotted by Fiji software in (B).


assembly of V - ATPase V1 - V0 holoenzymes in WT RPE1 over - expressing α - tubulin deacetylase HDAC6 or EGFP as a control . Representative immunoblots for ATP6V0D , ATP6V1A , ATP6V1C1 , LAMP1 GAPDH are shown in ( H ) .

O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O

The assembly of V-ATPase V1-V0 holoenzymes in WT RPE1 cells over-expressing the α-tubulin deacetylase HDAC6 or EGFP as a control. Representative immunoblots for ATP6V0D, ATP6V1A, ATP6V1C1, LAMP1 and GAPDH are shown in (H).


( M - O ) Loss of NDST3 enhances microtubule acetylation colocalization of lysosomes with V - ATPase V1 subunits . WT NDST3 KO PRE1 stably expressing LAMP1 - GFP were fixed analyzed by immunofluorescence with antibodies against ATP6V1C1 Ac - α - tubulin . relative of Ac - α - tubulin ( N ) ( n = 75 in WT group , n = 79 in NDST3 KO group , * * * * P < 0 . 0001 ) colocalization of LAMP1 - GFP with ATP6V1C1 ( O ) ( n = 75 in WT group , n = 79 in NDST3 KO group , * * * * P < 0 . 0001 ) are quantified .

O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(M-O) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V-ATPase V1 subunits. WT and NDST3 KO PRE1 cells stably expressing LAMP1-GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac-α-tubulin. The relative levels of Ac-α-tubulin (N) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) and the colocalization of LAMP1-GFP with ATP6V1C1 (O) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) are quantified.