( D ) The total level of p62 and GFP - LC3 ( normalized to control cells ) and their degree of colocalization ( % of total ) in control and Tsg101 - and Vps24 - depleted cells were quantified using the Zeiss LSM 510 Meta software . 30 cells from three independent experiments were used for quantification . Error bars = SEM .

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(D) The total level of p62 and GFP-LC3 (normalized to control cells) and their degree of colocalization (% of total) in control and Tsg101- and Vps24-depleted cells were quantified using the Zeiss LSM 510 Meta software. 30 cells from three independent experiments were used for quantification. Error bars = SEM.


( E ) Western blot showing accumulation of LC3 - II and p62 in HeLa cells depleted of Vps24 and Tsg101 . p62 also accumulates in the insoluble fraction ( bottom picture ) , indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101 .

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(E) Western blot showing accumulation of LC3-II and p62 in HeLa cells depleted of Vps24 and Tsg101. p62 also accumulates in the insoluble fraction (bottom picture), indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101.


( F ) Western blot showing that LC3 - II levels were similar in control and Vps24 - depleted cells treated with Bafilomycin A .

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(F) Western blot showing that LC3-II levels were similar in control and Vps24-depleted cells treated with Bafilomycin A.


Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24 . ( A ) The double - tagged LC3 protein ( mCherry - GFP - LC3 ) will emit yellow ( green merged with red ) fluorescence in non - acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures . ( B - F ) HeLa cells were transfected with control ( B ) , Tsg101 ( C ) , or Vps24 ( D ) siRNA for 4 d and then with mCherry - GFP - LC3 for another 24 h before confocal microscopy analysis . Bar , 5

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Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24. (A) The double-tagged LC3 protein (mCherry-GFP-LC3) will emit yellow (green merged with red) fluorescence in non-acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures. (B-F) HeLa cells were transfected with control (B), Tsg101 (C), or Vps24 (D) siRNA for 4 d and then with mCherry-GFP-LC3 for another 24 h before confocal microscopy analysis. Bar, 5 μm


( E ) The mCherry - LC3 ( red ) signal was quantified in ∼ 30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry - GFP - LC3 . Error bar = SEM .

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(E) The mCherry-LC3 (red) signal was quantified in ∼30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry-GFP-LC3. Error bar = SEM.


( F ) Degradation of [ 14C ] - valine - labeled proteins in cells incubated in complete medium ( control ) , serum - and amino acid - deficient medium ( starve ) , or starvation media in the presence of 3 - methyl adenine ( 3 - MA ) to inhibit autophagic degradation . Average degradation per hour from four experiments done in duplicates ± 1 SEM are shown .

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(F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.


Autophagosomes and amphisomes do form in ESCRT - depleted cells . HeLa cells stably expressing GFP - LC3 were transfected with control ( A ) , Tsg101 ( B , D , and E ) , or Vps24 ( C ) siRNA and processed for immuno - EM analysis . Cryosections were incubated with antibodies against GFP ( 15 - nm gold ) and LBPA ( 10 - nm gold ) . Amphisomes ( Am ) , characterized by their content of electron - dense LC3 - positive areas and intraluminal vesicles , were observed in both control ( A ) and ESCRT - depleted cells ( B and C ) . ( D ) In the Tsg101 - depleted cells we found large clusters of more typical autophagosomes ( Au ) and ( E ) clusters of double - membrane structures , consisting of autophagosomes and tubular structures which might represent phagophores , all labeling strongly for GFP - LC3 . We never observed similar clusters in control cells and very rarely in Vps24 - depleted cells . Bars , 200 nm . ( F ) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24 . Approximately 20 cells were included per siRNA depletion per experiment ( n = 3 ) . Error bars = SEM .

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Autophagosomes and amphisomes do form in ESCRT-depleted cells. HeLa cells stably expressing GFP-LC3 were transfected with control (A), Tsg101 (B, D, and E), or Vps24 (C) siRNA and processed for immuno-EM analysis. Cryosections were incubated with antibodies against GFP (15-nm gold) and LBPA (10-nm gold). Amphisomes (Am), characterized by their content of electron-dense LC3-positive areas and intraluminal vesicles, were observed in both control (A) and ESCRT-depleted cells (B and C). (D) In the Tsg101-depleted cells we found large clusters of more typical autophagosomes (Au) and (E) clusters of double-membrane structures, consisting of autophagosomes and tubular structures which might represent phagophores, all labeling strongly for GFP-LC3. We never observed similar clusters in control cells and very rarely in Vps24-depleted cells. Bars, 200 nm. (F) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24. Approximately 20 cells were included per siRNA depletion per experiment (n = 3). Error bars = SEM.


Immuno - EM of p62 - positive structures in Vps24 - and Tsg101 - depleted cells . Cryosections of HeLa cells transfected with Vps24 ( A , C , and E ) or Tsg101 ( B , D , and F ) siRNA were incubated with antibodies against p62 ( 15 - nm protein A gold , 10 - nm in D ) . Membrane - free p62 - positive aggregates ( C and D ) or p62 - positive structures contained within dense clusters of vesicular - tubular elements ( A and B ) were detected . Vesicles with early endosomal morphology ( EE ) or multivesicular appearance ( MVB ) associated with these clusters . We also observed p62 labeling in electron - dense structures sequestered within amphisomes in cell depleted of Vps24 ( E ) or Tsg101 ( F ) . The amphisomes typically consist of an electron - dense p62 positive body ( E and F , arrowhead ) , and a multivesicular endosomal structure ( E and F , arrow ) . Bars , 200 nm .

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Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells. Cryosections of HeLa cells transfected with Vps24 (A, C, and E) or Tsg101 (B, D, and F) siRNA were incubated with antibodies against p62 (15-nm protein A gold, 10-nm in D). Membrane-free p62-positive aggregates (C and D) or p62-positive structures contained within dense clusters of vesicular-tubular elements (A and B) were detected. Vesicles with early endosomal morphology (EE) or multivesicular appearance (MVB) associated with these clusters. We also observed p62 labeling in electron-dense structures sequestered within amphisomes in cell depleted of Vps24 (E) or Tsg101 (F). The amphisomes typically consist of an electron-dense p62 positive body (E and F, arrowhead), and a multivesicular endosomal structure (E and F, arrow). Bars, 200 nm.


Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants . HeLa cells transfected with myc - tagged wild - type CHMP2B ( A ) , CHMP2BIntron5 ( B ) , or CHMP2BΔ10 ( C ) were labeled with antibodies against c - Myc ( red ) , ubiquitin ( green ) , and p62 ( blue ) and analyzed by confocal microscopy . Single channel images in black and white are shown . Bars , 10 μm .

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Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants. HeLa cells transfected with myc-tagged wild-type CHMP2B (A), CHMP2BIntron5 (B), or CHMP2BΔ10 (C) were labeled with antibodies against c-Myc (red), ubiquitin (green), and p62 (blue) and analyzed by confocal microscopy. Single channel images in black and white are shown. Bars,10 μm.


( D ) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control ( mock transfected ) cells . Error bars = SEM .

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(D) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control (mock transfected) cells. Error bars = SEM.


( E ) Western blot analysis showing accumulation of p62 in cells transfected with myc - CHMP2BIntron5 compared with mock - transfected cells ( control ) and cells transfected with

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(E) Western blot analysis showing accumulation of p62 in cells transfected with myc-CHMP2BIntron5 compared with mock-transfected cells (control) and cells transfected with CHMP2Bwt


( F ) Immuno - EM showing p62 - positive aggregates containing dense clusters of vesicular - tubular structures in cells transfected with myc - CHMP2BIntron5 . Bar , 200 nm .

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(F) Immuno-EM showing p62-positive aggregates containing dense clusters of vesicular-tubular structures in cells transfected with myc-CHMP2BIntron5. Bar, 200 nm.


TDP - 43 accumulates in cytoplasmic ubiquitin - positive structures in Tsg101 - and Vps24 - depleted cells . HeLa cells transfected with control ( A ) , Tsg101 ( B ) , or Vps24 ( C ) siRNA were fixed , permeabilized , and stained with antibodies against TDP - 43 ( green ) , ubiquitin ( red ) , and p62 ( blue ) . Single channel images in black and white are shown . Colocalization of all proteins is indicated in white in the merged picture . Bars , 10 μm .

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TDP-43 accumulates in cytoplasmic ubiquitin-positive structures in Tsg101- and Vps24-depleted cells. HeLa cells transfected with control (A), Tsg101 (B), or Vps24 (C) siRNA were fixed, permeabilized, and stained with antibodies against TDP-43 (green), ubiquitin (red), and p62 (blue). Single channel images in black and white are shown. Colocalization of all proteins is indicated in white in the merged picture. Bars, 10 μm.


Vps24 is required for efficient clearance of Htt inclusions . HeLa cells expressing HttQ65 - mCFP ( A ) or HttQ103 - mCFP ( B ) and N2a cells expressing HttQ103 - mCFP ( C ) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65 / Q103 - mCFP . Vps24 depletion was determined by Western blot analysis . ( A and C ) Clearance of SDS - insoluble Htt inclusions was analyzed by filter - trap assays . ( B ) Alternatively , aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox . 300 cells from three independent experiments were counted for each condition . Error bars = SD .

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Vps24 is required for efficient clearance of Htt inclusions. HeLa cells expressing HttQ65-mCFP (A) or HttQ103-mCFP (B) and N2a cells expressing HttQ103-mCFP (C) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65/Q103-mCFP. Vps24 depletion was determined by Western blot analysis. (A and C) Clearance of SDS-insoluble Htt inclusions was analyzed by filter-trap assays. (B) Alternatively, aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox. 300 cells from three independent experiments were counted for each condition. Error bars = SD.


( A ) Mitochondrial morphology in HeLa cells depleted of Sept2 , Sept7 and Sept9 . Mitochondria were labeled with mitotracker ( green ) . Scale bar : 10 µm . The inset represents a twofold enlargement . ( B ) Quantification of mean mitochondrial length in mock treated cells and in cells depleted of Sept2 , Sept7 and Sept9 . n > 250 individual mitochondria from three independent experiments .

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(A) Mitochondrial morphology in HeLa cells depleted of Sept2, Sept7 and Sept9. Mitochondria were labeled with mitotracker (green). Scale bar: 10 µm. The inset represents a twofold enlargement. (B) Quantification of mean mitochondrial length in mock treated cells and in cells depleted of Sept2, Sept7 and Sept9. n>250 individual mitochondria from three independent experiments.


( C ) Mitochondrial morphology in mock treated cells or HeLa cells depleted of Sept2 and then transfected with empty vector , siRNA - resistant HA - tagged Sept2 ( Sept2 - rescue ) or Sept7 ( Sept7 - rescue ) . Mitochondria were labeled with mitotracker ( shown in green ) , asterisks mark transfected cells . Scale bar : 10 µm , insets are twofold enlargements . ( D ) Quantification of mean mitochondrial length in mock treated cells , in cells depleted of Sept2 or in cells depleted of Sept2 and transfected with siRNA - resistant Sept2 ( Sept2 - rescue ) or Sept7 ( Sept7 - rescue ) . n > 200 individual mitochondria from two independent experiments .

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(C) Mitochondrial morphology in mock treated cells or HeLa cells depleted of Sept2 and then transfected with empty vector, siRNA-resistant HA-tagged Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). Mitochondria were labeled with mitotracker (shown in green), asterisks mark transfected cells. Scale bar: 10 µm, insets are twofold enlargements. (D) Quantification of mean mitochondrial length in mock treated cells, in cells depleted of Sept2 or in cells depleted of Sept2 and transfected with siRNA-resistant Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). n>200 individual mitochondria from two independent experiments.


( E ) Mock treated or Sept2 depleted U2OS cells stained for Sept2 ( green ) and the Golgi apparatus ( GM130 , red ) , the ER ( Sec61b - GFP , displayed in red ) or peroxisomes ( PMP70 , red ) . ( F ) Quantification of the mean Golgi area ( n = 40 cells , three independent experiments ) ( G ) Quantification showing the percentage of ER traversing well - resolved mitochondrial tubules ( n = 15 cells from three independent experiments ) . ( H ) Quantification of average peroxisome counts per cell ( n = 40 cells from three independent experiments ) . Scale bar : 10 µm , insets are enlarged twofold .

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(E) Mock treated or Sept2 depleted U2OS cells stained for Sept2 (green) and the Golgi apparatus (GM130, red), the ER (Sec61b-GFP, displayed in red) or peroxisomes (PMP70, red). (F) Quantification of the mean Golgi area (n=40 cells, three independent experiments) (G) Quantification showing the percentage of ER traversing well-resolved mitochondrial tubules (n=15 cells from three independent experiments). (H) Quantification of average peroxisome counts per cell (n=40 cells from three independent experiments). Scale bar: 10 µm, insets are enlarged twofold.


( A ) Live cell imaging of mock treated or Sept2 depleted U2OS cells analyzed with the mito - PA - GFP mitochondrial fusion assay . Mito - PA - GFP is photoactivated in the indicated ROI and the decrease in fluorescence is followed in the same ROI , correcting for cell movement ( B ) Quantification of mitochondrial fusion rates in mock treated or Sept2 depleted cells expressed as a means of three independent experiments SEM . ( C ) Quantification of fission rates in mock and Sept2 depleted cells . n = 116 fission events for Sept2 siRNA , n = 83 fission events for mock , two independent experiments .

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(A) Live cell imaging of mock treated or Sept2 depleted U2OS cells analyzed with the mito-PA-GFP mitochondrial fusion assay. Mito-PA-GFP is photoactivated in the indicated ROI and the decrease in fluorescence is followed in the same ROI, correcting for cell movement (B) Quantification of mitochondrial fusion rates in mock treated or Sept2 depleted cells expressed as a means of three independent experiments SEM. (C) Quantification of fission rates in mock and Sept2 depleted cells. n=116 fission events for Sept2 siRNA, n=83 fission events for mock, two independent experiments.


( D ) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane ( OM - GFP ) , showing delayed FCCP - induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells . White arrowheads point at fission examples , yellow arrowheads to looping . Scale bar : 10 µm , insets are enlarged twofold . ( E ) Quantification of mitochondrial elongation ( long / short axis ) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells , mean of four independent experiments SEM .

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(D) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane (OM-GFP), showing delayed FCCP-induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells. White arrowheads point at fission examples, yellow arrowheads to looping. Scale bar: 10 µm, insets are enlarged twofold. (E) Quantification of mitochondrial elongation (long /short axis) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells, mean of four independent experiments SEM.


( A ) Immunofluorescence microscopy analysis of endogenous Sept2 ( green ) and mitochondria ( Tom20 , red ) in HeLa cells . The insets show a twofold enlargement with arrowheads highlighting the presence of small Sept2 structures on mitochondria . Scale bar 10 µm . ( B ) 3D rendering of confocal microscopy sections showing Sept2 at mitochondrial constrictions ( white arrowheads ) and constricted mitochondria that are not marked by Sept2 ( yellow arrowhead ) Scale bar 2 µm .

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(A) Immunofluorescence microscopy analysis of endogenous Sept2 (green) and mitochondria (Tom20, red) in HeLa cells. The insets show a twofold enlargement with arrowheads highlighting the presence of small Sept2 structures on mitochondria. Scale bar 10 µm. (B) 3D rendering of confocal microscopy sections showing Sept2 at mitochondrial constrictions (white arrowheads) and constricted mitochondria that are not marked by Sept2 (yellow arrowhead) Scale bar 2 µm.


( C ) CoDiM super - resolution imaging of endogenous Sept2 . Arrowheads highlight the presence of Sept2 at mitochondrial constrictions . Insets show a threefold enlargement Scale bar 10 μm .

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(C) CoDiM super-resolution imaging of endogenous Sept2. Arrowheads highlight the presence of Sept2 at mitochondrial constrictions. Insets show a threefold enlargement Scale bar 10 μm.


( D ) Still images from a live cell imaging experiment showing Sept2 ( green ) accumulation during two mitochondrial fission events ( mitotracker orange , red ) , highlighted by yellow ( first fission ) or green arrowheads ( second fission ) . Scale bar 2 µm .

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(D) Still images from a live cell imaging experiment showing Sept2 (green) accumulation during two mitochondrial fission events (mitotracker orange, red), highlighted by yellow (first fission) or green arrowheads (second fission). Scale bar 2 µm.


( A ) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co - immunoprecipitates with Sept2 . The asterisks mark the IgG heavy chains .

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(A) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co-immunoprecipitates with Sept2. The asterisks mark the IgG heavy chains.


( B ) Elyra SIM image of U2OS cells stained for Sept2 ( green ) , mitochondria ( GFP targeted to the mitochondrial outer membrane ( OM - GFP ) , shown in red ) , Drp1 ( blue ) . Yellow arrows point at tetraspeck beads used for image alignment . Insets represent sevenfold enlargements . White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria . Scale bar : 10 µm , insets are enlarged fourfold .

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(B) Elyra SIM image of U2OS cells stained for Sept2 (green), mitochondria (GFP targeted to the mitochondrial outer membrane (OM-GFP), shown in red), Drp1 (blue). Yellow arrows point at tetraspeck beads used for image alignment. Insets represent sevenfold enlargements. White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria. Scale bar: 10 µm, insets are enlarged fourfold.


( C ) GST pulldown of recombinant Sept2 with GST - Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1 .

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(C) GST pulldown of recombinant Sept2 with GST-Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1.


( D ) Sept2 immunoprecipitation from mock or CCCP - treated HeLa cells probed for Drp1 and Sept2 . ( E ) Quantification of two independent experiments showing stimulated Drp1 co - immunoprecipitation with Sept2 in FCCP - treated cells .

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(D) Sept2 immunoprecipitation from mock or CCCP-treated HeLa cells probed for Drp1 and Sept2. (E) Quantification of two independent experiments showing stimulated Drp1 co-immunoprecipitation with Sept2 in FCCP-treated cells.


( F ) Sept2 immunoprecipitation from mock or Mdivi - 1 - treated HeLa cells probed for Drp1 and Sept2 . ( G ) Quantification of two independent experiments showing decreased Drp1 co - immunoprecipitation with Sept2 upon Mdivi - 1 treatment .

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

(F) Sept2 immunoprecipitation from mock or Mdivi-1-treated HeLa cells probed for Drp1 and Sept2. (G) Quantification of two independent experiments showing decreased Drp1 co-immunoprecipitation with Sept2 upon Mdivi-1 treatment.


( H ) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 ( green , Sept2 - YFP ) , Drp1 ( red , Drp1 - mCherry ) and mitochondria ( blue , mitotracker DeepRed ) during mitochondrial fission . Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission ( yellow arrowheads ) . Scale bar : 2 µm .

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

(H) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 (green, Sept2-YFP), Drp1 (red, Drp1-mCherry) and mitochondria (blue, mitotracker DeepRed) during mitochondrial fission. Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission (yellow arrowheads). Scale bar: 2 µm.


( A ) Immunofluorescence microscopy of Sept2 ( blue ) , mitochondria ( Tom20 , red ) and Drp1 ( green ) in mock treated and Sept2 depleted cells . Scale bar : 10 µm , insets are enlarged fourfold . ( B ) Quantification of mitochondria - associated Drp1 in mock treated and Sept2 depleted cells ( mean of 3 independent experiments , n = 44 - 51 cells ) ( C ) Average distance between mitochondria - associated Drp1 oligomers ( representative experiment with n > 25 individual mitochondria ) .

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

(A) Immunofluorescence microscopy of Sept2 (blue), mitochondria (Tom20, red) and Drp1 (green) in mock treated and Sept2 depleted cells. Scale bar: 10 µm, insets are enlarged fourfold. (B) Quantification of mitochondria-associated Drp1 in mock treated and Sept2 depleted cells (mean of 3 independent experiments, n=44-51 cells) (C) Average distance between mitochondria-associated Drp1 oligomers (representative experiment with n>25 individual mitochondria).


( D ) HeLa cytosol and crude mitochondrial fractions prepared from mock and Sept2 siRNA treated cells . The samples were analysed by western blotting with indicated antibodies , and show a decrease in mitochondria - associated Drp1 in Sept2 depleted cells .

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

(D) HeLa cytosol and crude mitochondrial fractions prepared from mock and Sept2 siRNA treated cells. The samples were analysed by western blotting with indicated antibodies, and show a decrease in mitochondria-associated Drp1 in Sept2 depleted cells.


( A ) Immunofluorescence microscopy analysis of P - MLC ( green ) recruitment to mitochondria ( red , Tom20 ) in mock treated and Sept2 depleted HeLa cells . Insets show twofold enlargements . Scale bar : 10 µm . ( B ) Colocalization of P - MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells .

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

(A) Immunofluorescence microscopy analysis of P-MLC (green) recruitment to mitochondria (red, Tom20) in mock treated and Sept2 depleted HeLa cells. Insets show twofold enlargements. Scale bar: 10 µm. (B) Colocalization of P-MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells.


( C ) Mock treated or Sept2 depleted Drp1 - / - MEF cells were labeled with mitotracker orange ( red ) , treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin ( green ) . Insets are enlarged twofold .

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

(C) Mock treated or Sept2 depleted Drp1 -/- MEF cells were labeled with mitotracker orange (red), treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin (green). Insets are enlarged twofold.


( D ) Immunofluorescence microscopy analysis of the actin binding proteins Arp3 and cofilin ( green ) , colocalized with cytocrome c ( red ) and cortactin ( green ) , colocalized with Hsp60 ( red ) in mock treated or Sept2 depleted Drp1 - / - MEF cells . Scale bars : 10 µm .

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

(D) Immunofluorescence microscopy analysis of the actin binding proteins Arp3 and cofilin (green), colocalized with cytocrome c (red) and cortactin (green), colocalized with Hsp60 (red) in mock treated or Sept2 depleted Drp1 -/- MEF cells. Scale bars: 10 µm.


( E ) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 - / - MEF cells and analyzed by western blotting with the indicated antibodies .

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

(E) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 -/- MEF cells and analyzed by western blotting with the indicated antibodies.


( F ) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation . Immunoprecipitates were analyzed by western blot for Drp1 and Sept2 , showing that Drp1 co - immunoprecipitation with Sept2 is not affected by cytochalasin D treatment .

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

(F) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation. Immunoprecipitates were analyzed by western blot for Drp1 and Sept2, showing that Drp1 co-immunoprecipitation with Sept2 is not affected by cytochalasin D treatment.


( A ) Live imaging of C . elegans expressing TOM70 : : GFP in body wall muscle cells . Depletion of the UNC - 59 or UNC - 61 led to an increase in mitochondrial network connectivity as shown by quantification in ( B ) . Insets represent a twofold magnification . Scale bar : 10 µm .

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

(A) Live imaging of C. elegans expressing TOM70::GFP in body wall muscle cells. Depletion of the UNC-59 or UNC-61 led to an increase in mitochondrial network connectivity as shown by quantification in (B). Insets represent a twofold magnification. Scale bar: 10 µm.


( C ) Live imaging of C . elegans body wall muscle cells expressing TOM70 : : GFP . Depletion of DRP - 1 or UNC - 61 caused an increase in mitochondrial elongation and network connectivity , which did not increase upon combination of DRP - 1 and UNC - 61 . Insets represent a twofold magnification . Scale bar : 10 µm .

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

(C) Live imaging of C. elegans body wall muscle cells expressing TOM70::GFP. Depletion of DRP-1 or UNC-61 caused an increase in mitochondrial elongation and network connectivity, which did not increase upon combination of DRP-1 and UNC-61. Insets represent a twofold magnification. Scale bar: 10 µm.


A ) Caspase 3 / 7 activation ± 16 - hour treatment with varying concentrations of BAM15 , DNP , or FCCP ( N = 3 per condition ) . 5 μM : BAM15 vs . DNP ( P = 0 . 030 ) . 10 μM : BAM15 vs . DNP ( P = 0 . 013 ) . 20 μM : BAM15 vs . DNP ( P = 0 . 0005 ) , BAM15 vs . FCCP ( P = 0 . 036 ) . 40 μM : BAM15 vs . DNP ( P = 0 . 018 ) , BAM15 vs . FCCP ( P = 0 . 021 ) . 80 μM : BAM15 vs . DNP ( P = 0 . 018 ) , DNP vs . FCCP ( P = 0 . 004 ) .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Caspase 3/7 activation ± 16-hour treatment with varying concentrations of BAM15, DNP, or FCCP (N=3 per condition). 5 μM: BAM15 vs. DNP (P=0.030). 10 μM: BAM15 vs. DNP (P=0.013). 20 μM: BAM15 vs. DNP (P=0.0005), BAM15 vs. FCCP (P=0.036). 40 μM: BAM15 vs. DNP (P=0.018), BAM15 vs. FCCP (P=0.021). 80 μM: BAM15 vs. DNP (P=0.018), DNP vs. FCCP (P=0.004).


B ) The rate of oxygen consumption ( OCR ) , ( C ) ratio of oxygen consumption to extracellular acidification ( OCR / ECAR ) , ollowing acute injection of 1 μM BAM15 , DNP , or FCCP ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) ( N = 6 per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The rate of oxygen consumption (OCR), (C) ratio of oxygen consumption to extracellular acidification (OCR/ECAR), ollowing acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).


D ) maximal rate of OCR ( BAM15 vs . DNP , P < 0 . 0001 ; DNP vs . FCCP ( P < 0 . 0001 ) ( E ) time to maximal uncoupling rate ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) , and ( F ) mitochondrial respiratory half - life following acute injection of 1 μM BAM15 , DNP , or FCCP ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) ( N = 6 per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) maximal rate of OCR (BAM15 vs. DNP, P<0.0001; DNP vs. FCCP (P<0.0001) (E) time to maximal uncoupling rate (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001), and (F) mitochondrial respiratory half-life following acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).


G ) Representative respirometry plot of intact cells ± 16 - hour treatment with 20 μM BAM15 sequentially injected with oligomycin , FCCP , and a cocktail of rotenone and antimycin a ( N = 5 per condition )

O O O O O O O O O O O O O O O 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) Representative respirometry plot of intact cells ± 16-hour treatment with 20 μM BAM15 sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)


H ) basal ( P = 0 . 002 ) and ATP - linked respiration , proton leak ( P = 0 . 0003 ) , maximal uncoupling , spare respiratory capacity ( P < 0 . 0001 ) , and non - mitochondrial respiration ( N = 5 per condition ) .

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

H) basal (P=0.002) and ATP-linked respiration, proton leak (P=0.0003), maximal uncoupling, spare respiratory capacity (P<0.0001), and non-mitochondrial respiration (N=5 per condition).


I ) Assessment of routine ( R ) oxygen flux ( P = 0 . 030 ) , leak ( L ) ( P = 0 . 010 ) , OXPHOS ( P ) and ETC ( E ) capacity in permeabilized C2C12 cells ± 16 - hour treatment with BAM15 ( N = 4 per condition ) . PM : pyruvate and malate , ADP : adenosine diphosphate , GS : glutamate and succinate , Tm : tetramethyl - p - phenylenediamine .

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

I) Assessment of routine (R) oxygen flux (P=0.030), leak (L) (P=0.010), OXPHOS (P) and ETC (E) capacity in permeabilized C2C12 cells ± 16-hour treatment with BAM15 (N=4 per condition). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine.


( J ) Citrate synthase activity ± 16 - hour treatment with 20 μM BAM15 ( N = 10 per condition ) . ( K ) mtDNA ( COX2 : 18S ) ± 16 - hour treatment with 20 μM BAM15 ( N = 6 per condition ) .

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

(J) Citrate synthase activity ± 16-hour treatment with 20 μM BAM15 (N=10 per condition). (K) mtDNA (COX2:18S) ± 16-hour treatment with 20 μM BAM15 (N=6 per condition).


( A ) Heat map visualization of top 30 differentially regulated mRNA transcripts . Color represents significant down - ( blue ) or up - regulated ( red ) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O 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) Heat map visualization of top 30 differentially regulated mRNA transcripts. Color represents significant down- (blue) or up-regulated (red) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate.


- D ( A ) Representative immunoblots and ( B ) quantitative analysis of AKT ( 1 μM insulin : P = 0 . 038 ) , ( C ) AS160 ( - insulin : P = 0 . 036 , 1 μM insulin : P = 0 . 0038 ) , and ( D ) AMPK ( - insulin : P = 0 . 018 ) phosphorylation ± 16 hour BAM15 treatment and 15 minute insulin stimulation ( N = 3 per condition ) in C2C12 myotubes .

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

-D (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P=0.038), (C) AS160 (- insulin: P=0.036, 1 μM insulin: P=0.0038), and (D) AMPK (- insulin: P=0.018) phosphorylation ± 16 hour BAM15 treatment and 15 minute insulin stimulation (N=3 per condition) in C2C12 myotubes.


, F ( E ) Representative immunoblots and quantitative analysis of ( F ) plasma membrane GLUT4 translocation ( P = 0 . 0139 ) ± 16 - hour BAM15 treatment ( N = 3 per condition ) in C2C12 myotubes .

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

, F (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation (P=0.0139) ± 16-hour BAM15 treatment (N=3 per condition) in C2C12 myotubes.


G ) [ 3 - 3H ] glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation ( N = 4 per condition ; vehicle vs . vehicle + insulin : P = 0 . 0016 , vehicle vs . BAM15 : P < 0 . 0001 , vehicle vs . BAM15 + insulin : P < 0 . 0001 , vehicle + insulin vs . BAM15 + insulin : P = 0 . 0018 , BAM15 vs . BAM15 + insulin : P = 0 . 0024 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) [3-3H]glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation (N=4 per condition; vehicle vs. vehicle + insulin: P=0.0016, vehicle vs. BAM15: P<0.0001, vehicle vs. BAM15 + insulin: P<0.0001, vehicle + insulin vs. BAM15 + insulin: P=0.0018, BAM15 vs. BAM15 + insulin: P=0.0024)


H ) [ 1 - 14C ] palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate ( N = 6 per condition ; no glucose / pyruvate : P < 0 . 0001 ) in C2C12 myotubes .

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

H) [1-14C]palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate (N=6 per condition; no glucose/pyruvate: P<0.0001) in C2C12 myotubes.


I ) Representative immunoblot and quantitative analysis ( N = 5 per condition ; P < 0 . 0001 ) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes .

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

I) Representative immunoblot and quantitative analysis (N=5 per condition; P<0.0001) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes.


J ) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin , FCCP , and a cocktail of rotenone and antimycin a ( N = 5 per condition )

O O O O O O O O O O O O O O O O O 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) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)


K ) basal and ATP - linked respiration , proton leak , maximal uncoupling , and spare respiratory capacity ( N = 5 per condition ) . Basal : shEV vs . shEV + BAM15 ( P = 0 . 0317 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) . ATP : shEV vs . shAMPK + BAM15 ( P = 0 . 0026 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P = 0 . 0003 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 040 ) . Proton leak : shEV vs . shEV + BAM15 ( P = 0 . 0052 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 0394 ) . Maximal : shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) . Spare Capacity : shEV vs . shAMPK ( P = 0 . 0028 ) , shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) .

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

K) basal and ATP-linked respiration, proton leak, maximal uncoupling, and spare respiratory capacity (N=5 per condition). Basal: shEV vs. shEV + BAM15 (P=0.0317), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001). ATP: shEV vs. shAMPK + BAM15 (P=0.0026), shEV + BAM15 vs. shAMPK + BAM15 (P=0.0003), shAMPK vs. shAMPK + BAM15 (P=0.040). Proton leak: shEV vs. shEV + BAM15 (P=0.0052), shAMPK vs. shAMPK + BAM15 (P=0.0394). Maximal: shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001). Spare Capacity: shEV vs. shAMPK (P=0.0028), shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001).


L ) [ 3 - 3H ] glucose uptake ± shAMPK , 16 hour BAM15 treatment , and 30 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) . No insulin : shEV vs . shEV + BAM15 ( P = 0 . 0001 ) . Insulin : shEV vs . shEV + BAM15 ( P = 0 . 0142 ) .

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

L) [3-3H]glucose uptake ± shAMPK, 16 hour BAM15 treatment, and 30 minute insulin stimulation in C2C12 myotubes (N=3 per condition). No insulin: shEV vs. shEV + BAM15 (P=0.0001). Insulin: shEV vs. shEV + BAM15 (P=0.0142).


- O ( M ) Representative immunoblots and quantitative analysis of ( N ) AKT ( shEV + insulin : P = 0 . 0128 , shAMPK - insulin : P = 0 . 0018 , shAMPK + insulin , P = 0 . 0124 ) , and ( O ) AS160 ( shEV - insulin : P = 0 . 0283 , shEV + insulin : P = 0 . 0137 ) phosphorylation ± shAMPK , 16 hour BAM15 treatment , and 15 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) .

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

-O (M) Representative immunoblots and quantitative analysis of (N) AKT (shEV + insulin: P=0.0128, shAMPK - insulin: P=0.0018, shAMPK + insulin, P=0.0124), and (O) AS160 (shEV - insulin: P=0.0283, shEV + insulin: P=0.0137) phosphorylation ± shAMPK, 16 hour BAM15 treatment, and 15 minute insulin stimulation in C2C12 myotubes (N=3 per condition).


( A ) Serum concentrations of BAM15 over a 24 - hour period ( N = 3 animals per time point )

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

(A) Serum concentrations of BAM15 over a 24-hour period (N=3 animals per time point)


( B ) peak tissue concentrations of BAM15 ( N = 6 per tissue ) .

(B) peak tissue concentrations of BAM15 (N=6 per tissue).


C , D ( C ) Daily body weight ( day 8 : P = 0 . 0454 , day 9 : P = 0 . 0353 , day 10 : P = 0 . 0251 , day 11 : P = 0 . 0131 , day 12 : 0 . 0038 , day 13 : P = 0 . 0008 , day 14 : P = 0 . 0005 , day 15 : P = 0 . 0003 , day 16 - 21 : P < 0 . 0001 ) and ( D ) cumulative food intake over the 3 - week treatment in CTRL and BAM15 treated animals ( N = 16 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Daily body weight (day 8: P=0.0454, day 9: P=0.0353, day 10: P=0.0251, day 11: P=0.0131, day 12: 0.0038, day 13: P=0.0008, day 14: P=0.0005, day 15: P=0.0003, day 16-21: P<0.0001) and (D) cumulative food intake over the 3-week treatment in CTRL and BAM15 treated animals (N=16 per group).


( E ) Whole body temperature before ( pre ) , one hour ( 1 hr Post ) , and 24 hours ( 24 hrs Post ) after intraperitoneal injection of corn oil , vehicle ( corn oil and saline ) , 0 . 1 , 0 . 5 , and , 1 mg BAM15 per kg of body weight ( N = 11 per group ) ,

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

(E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group),


Whole body temperature ( F ) daily during chronic BAM15 treatment ( N = 8 per group ) .

Whole body temperature (F) daily during chronic BAM15 treatment (N=8 per group).


G , H ( G ) Change in fat mass ( % ) ( P < 0 . 0001 ) and ( H ) lean mass ( % ) ( P < 0 . 0001 ) from baseline to 3 - weeks in CTRL and BAM15 treated animals ( N = 8 per group ) .

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

G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group).


( I ) Fasting plasma glucose ( N = 8 per group ; P = 0 . 007 ) and ( J ) insulin ( N = 7 per group ; P = 0 . 0024 ) after 3 weeks of treatment in CTRL and BAM15 treated animals .

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

(I) Fasting plasma glucose (N=8 per group; P=0.007) and (J) insulin (N=7 per group; P=0.0024) after 3 weeks of treatment in CTRL and BAM15 treated animals.


( K ) Plasma glucose concentrations at 0 , 15 , 30 , 60 , 90 , and 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of body weight ; N = 8 per group )

(K) Plasma glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( L ) total area under the curve glucose after 3 - weeks in CTRL and BAM15 treated animals ( N = 8 per group ; P = 0 . 0106 ) .

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

(L) total area under the curve glucose after 3-weeks in CTRL and BAM15 treated animals (N=8 per group; P=0.0106).


M - O ( M ) Oxygen consumption rates ( N ) energy expenditure ( daily : P = 0 . 0357 , dark phase : P = 0 . 030 ) , and ( O ) respiratory exchange ratio ( daily : P = 0 . 0424 , dark phase : P = 0 . 0016 ) measured by continuous high - resolution indirect calorimetry after 3 - weeks of CTRL or BAM15 treatment ( N = 8 per group ) .

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

M-O (M) Oxygen consumption rates (N) energy expenditure (daily: P=0.0357, dark phase: P=0.030), and (O) respiratory exchange ratio (daily: P=0.0424, dark phase: P=0.0016) measured by continuous high-resolution indirect calorimetry after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( P ) Fecal energy density ( % recovered in feces ) after 3 - weeks of CTRL or BAM15 treatment ( N = 8 per group ) .

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

(P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( A ) Change in body weight ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P < 0 . 0001 ) , ( B ) fat mass ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0041 , BAM15 vs . CR : P = 0 . 043 ) , and ( C ) lean mass ( CTRL vs . BAM15 : P = 0 . 0014 , CTRL vs . CR : P < 0 . 0001 , BAM15 vs . CR : P = 0 . 0047 ) from baseline to 2 - weeks in CTRL , BAM15 , and CR treated

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Change in body weight (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P<0.0001), (B) fat mass (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0041, BAM15 vs. CR: P=0.043), and (C) lean mass (CTRL vs. BAM15: P=0.0014, CTRL vs. CR: P<0.0001, BAM15 vs. CR: P=0.0047) from baseline to 2-weeks in CTRL, BAM15, and CR treated animals


( D ) daily food intake ( CTRL vs . CR : P = 0 . 0092 , BAM15 vs . CR : P = 0 . 0175 ) in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) daily food intake (CTRL vs. CR: P=0.0092, BAM15 vs. CR: P=0.0175) in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( E ) Blood glucose concentrations at 0 , 15 , 30 , 60 , 90 , and 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of body weight ; N = 8 per group )

(E) Blood glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( F ) total area under the curve glucose ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P = 0 . 003 , BAM15 vs . CR : P = 0 . 0261 ) after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

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

(F) total area under the curve glucose (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P=0.003, BAM15 vs. CR: P=0.0261) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( G ) Plasma insulin concentrations at baseline ( PRE ) , and 0 ( CTRL vs . BAM15 : P = 0 . 0154 , CTRL vs . CR : P = 0 . 0425 ) and 120 minutes ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0211 , BAM15 vs . CR : P = 0 . 0264 ) after injection of glucose after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasma insulin concentrations at baseline (PRE), and 0 (CTRL vs. BAM15: P=0.0154, CTRL vs. CR: P=0.0425) and 120 minutes (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0211, BAM15 vs. CR: P=0.0264) after injection of glucose after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8)and


( H ) Homeostatic Model Assessment of Insulin Resistance ( HOMA - IR ) ( CTRL vs . BAM15 : P = 0 . 0259 , CTRL vs . CR : P = 0 . 040 ) after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

(H) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (CTRL vs. BAM15: P=0.0259, CTRL vs. CR: P=0.040) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( A ) Representative H & E stained liver , adipose tissue , and skeletal muscle sections from CTRL and BAM15 treated animals . Scale bars ( black ) = 100 μm .

(A) Representative H&E stained liver, adipose tissue, and skeletal muscle sections from CTRL and BAM15 treated animals. Scale bars (black) = 100 μm.


( B ) Representative immunohistochemical analysis of pancreatic insulin ( red ) content counterstained with diaminobenzidine ( blue ) . Scale bars ( black ) = 100 μm .

(B) Representative immunohistochemical analysis of pancreatic insulin (red) content counterstained with diaminobenzidine (blue). Scale bars (black) = 100 μm.


C - F ( C ) Quantitative analysis of liver steatosis ( μm2 ) ( N = 6 per group ; P < 0 . 0001 ) , ( D ) gastrocnemius muscle cross sectional area ( μm2 ) ( CTRL N = 7 , BAM15 N = 8 ) , ( E ) pancreatic insulin positive area ( % of total area ) ( N = 6 per group ; P = 0 . 0420 ) , ( F ) and β - cell mass ( N = 4 per group ; P = 0 . 0005 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F (C) Quantitative analysis of liver steatosis (μm2) (N=6 per group; P<0.0001), (D) gastrocnemius muscle cross sectional area (μm2)(CTRL N=7, BAM15 N=8), (E) pancreatic insulin positive area (% of total area) (N=6 per group; P=0.0420), (F) and β-cell mass (N=4 per group; P=0.0005).


( G - I ) Size ( area ; μm2 ) and distributional ( number ) analysis of ( G ) iWAT , ( H ) gWAT , and ( I ) BAT from CTRL and BAM15 treated animals ( N = 8 per group ) .

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

(G-I) Size (area; μm2) and distributional (number) analysis of (G) iWAT, (H) gWAT, and (I) BAT from CTRL and BAM15 treated animals (N=8 per group).


A - D ( A ) Plasma concentrations of non - esterified fatty acids ( NEFA ) ( CTRL N = 7 , BAM15 N = 8 , P = 0 . 0073 ) , ( B ) leptin ( N = 8 per group , P < 0 . 0001 ) , ( C ) GDF15 ( N = 7 per group , P = 0 . 035 ) , and ( D ) FGF21 ( N = 7 per group , P = 0 . 0026 ) after 3 - weeks of CTRL or BAM15 treatment .

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

A-D (A) Plasma concentrations of non-esterified fatty acids (NEFA) (CTRL N=7, BAM15 N=8, P=0.0073), (B) leptin (N=8 per group, P<0.0001), (C) GDF15 (N=7 per group, P=0.035), and (D) FGF21 (N=7 per group, P=0.0026) after 3-weeks of CTRL or BAM15 treatment.


E , F ( E ) Total ( basal : P = 0 . 015 ) and ( F ) complete ( basal : P = 0 . 0137 ) oxidation of 200 μM [ 1 - 14C ] palmitate to CO2 from mixed gastrocnemius muscle homogenates ( N = 7 per group ) .

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

E, F (E) Total (basal: P=0.015) and (F) complete (basal: P=0.0137) oxidation of 200 μM [1-14C]palmitate to CO2 from mixed gastrocnemius muscle homogenates (N=7 per group).


( G ) Routine ( R ) oxygen flux , leak ( L ) , OXPHOS ( P ) and ETC ( E ) capacity in permeabilized mixed gastrocnemius fibers ( N = 6 per group ) . PM : pyruvate and malate , ADP : adenosine diphosphate , GS : glutamate and succinate , Tm : tetramethyl - p - phenylenediamine , PalM : palmitoylcarnitine and malate , Oct : octanoylcarnitine , and Dur : duroquinol .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol.


( H ) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT , and mixed gastrocnemius

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

(H) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT, and mixed gastrocnemius muscle


( I - K ) Densitometric quantification ( N = 4 per group ; iWAT p - AMPKT172 : P = 0 . 0186 , iWAT p - ACCS79 : P = 0 . 0083 , mixed gastrocnemius p - AMPKT172 : P = 0 . 0286 ) .

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

(I-K) Densitometric quantification (N=4 per group; iWAT p-AMPKT172: P=0.0186, iWAT p-ACCS79: P=0.0083, mixed gastrocnemius p-AMPKT172: P=0.0286).


( L - N ) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT ( N = 7 per group ) . Srebf1 ( P < 0 . 0001 ) , Mlxipl ( P = 0 . 0018 ) , Scd1 ( P < 0 . 0001 ) , FASN ( P = 0 . 0009 ) , LIPE ( P = 0 . 0015 ) , Pnpla2 ( P < 0 . 0001 ) , PPARg ( P < 0 . 0001 ) .

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

(L-N) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT (N=7 per group). Srebf1 (P<0.0001), Mlxipl (P=0.0018), Scd1 (P<0.0001), FASN (P=0.0009), LIPE (P=0.0015), Pnpla2 (P<0.0001), PPARg (P<0.0001).


Bone marrow cells were polarized in the presence of 40 ng / ml GM - CSF and treated with 0 . 2 mM of the indicated compounds for 7 days . Gr1 - CD11b + cells were sorted and lysed for microarray . The hierarchical clustering was based on the different expression genes between BSA ( Con ) and oleate group .

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

Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group.


Signature genes involved in lipid metabolism , dendritic cell maturation , macrophage maturation , tumor associated macrophages ( TAMs ) phenotype , MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay . Data are expressed as mean ± SD from two to four independent experiments . Unpaired Student ' s two - tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups . * p < 0 . 05 ; * * p ≤ 0 .

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

Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01


Gr1 - CD11b + cells , polarized by the indicated treatment , were purified for mitochondrial respiration detection . The oxygen consumption rate ( OCR ) of these cells was monitored after the addition of oligomycin ( OA ) ( 1 µM ) , Carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA ) ( 0 . 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified .

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

Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


Forty µM etomoxir were added starting from day 0 of bone marrow polarization , followed by mitochondrial respiration assay by using the same amount of polarized Gr1 - CD11b + myeloid cells on day 7 . The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer .

Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer.


Exemplary plots of the proportion of CD206 + cells , the mean fluorescence intensity ( MFI ) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry .

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

Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry.


T cell proliferation assay was performed via co - culture with purified CD4 + T cells in the ratios of M ( myeloid cells ) : T ( T cells ) = 1 : 30 .

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

T cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30.


NO production from the co - culture supernatant was quantified by Griess reaction .

NO production from the co-culture supernatant was quantified by Griess reaction.


The oxygen consumption rate ( OCR ) of 1 × 105 purified Gr1 - CD11b + cells was monitored as described in Figure 2 . The basal OCR , basal ECAR ( extracellular acidification rate ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified .

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

The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


The polarization state was evaluated via the expression of CD206 .

The polarization state was evaluated via the expression of CD206.


T cell proliferation assay was performed employing co - culture with purified CD4 + T cell in the variant ratios .

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

T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios.


The lipid droplets accumulation was determined by BODIPY staining . The percentage of divided cells and proliferation index were calculated .

The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated.


Nitric oxide ( NO ) concentration in the co - culture supernatant was quantified by Griess reaction .

Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.


Bone marrow - derived myeloid cells were treated with BSA - control ( Con ) or oleate in the presence or absence of 10 nM rapamycin ( Rapa ) . The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression .

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

Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.


Purified Gr1 - CD11b + myeloid cells were co - cultured with CD4 + T cells for proliferation assay . The supernatant was collected for nitric oxide ( NO ) detection .

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

Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.


The oxygen consumption rate ( OCR ) of 1 × 105 differentiated myeloid cells were monitored after the addition of oligomycin ( OA ) ( 1 µM ) , the uncoupler carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA , 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , ATP production and maximal respiration based on the OCR value were quantified .

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

The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.


Mean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells as determined by flow

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

Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometry


Mean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells lysed for western blot .

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

Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.


Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206 + ( black ) and CD206 - ( grey ) cells .

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

Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.


Balb / c mice were inoculated with 5 × 105 CT26 tumor cells subcutaneously . Since day 6 , vehicle or DGAT inhibitors ( iDGAT ) were administrated via intra - tumoral injection once daily .

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

Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily.


At day 13 , tumor infiltrated immune cells were isolated and stained for CD206 and CD11b .

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

At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.


5 × 105 MCA205 were injected subcutaneously into C57BL / 6 mice . PBS , liposome control or iDGAT - liposome was injected into the peritoneum every other day starting at day 7 . Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis . Immune cells from spleen , tumor - draining lymph nodes ( TDLN ) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells .

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

5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.


Tumor infiltrating CD8 + T cells ( red ) were stained together with nuclei ( DAPI / blue ) after sacrificing the tumor bearing mice .

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Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.


1 × 106 CD14 + myeloid cells from healthy donors were polarized by 1 ng / ml GM - CSF in the presence of BSA - control ( C ) , 0 . 2 mM oleate ( O ) or 0 . 2 mM stearate ( S ) over a 6 - day period . The expression of CD206 , CD204 and CD38 was analyzed via flow cytometry .

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1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.


The level of lipid droplets was determined via BODIPY staining .

The level of lipid droplets was determined via BODIPY staining.


The oxygen consumption rate ( OCR ) of differentiated myeloid cells was monitored as described in Materials and Methods .

The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.


( D ) The purified CFSE - labeled autologous CD4 + T cells were co - cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay .

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(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.


Tumor tissue ( tumor ) as well as corresponding non - tumorous adjacent tissue ( control ) were collected from colorectal cancer patients and prepared for histopathology . The expression of CD68 ( red ) , CD206 ( green ) and ADRP ( blue ) indicate the lipid droplets in tumor infiltrating myeloid cells . Nuclei ( white ) were stained using DAPI . The absolute number of positive cells was quantified in 5 high - power fields ( hpf ) ( scale bar = 20 µm ) .

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Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).


A . Immunofluorescence microscopy of control , parental and patient fibroblasts labeled for LAMP - 1 . Patient fibroblasts ( VPS41S285P / R662 * and VPS41c . 1423 - 2A > G / R662 * ) show more LAMP - 1 puncta , which are distributed throughout the cell . ( A ' ) Quantification shows a significant increase in the number of LAMP - 1 positive compartments for both patients . > 15 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.


D . HeLaVPS41KO cells transfected with VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 constructs labeled for LAMP - 1 and V5 immunofluorescence microscopy . All VPS41 variants co - localize with LAMP - 1 , indicating that VPS41S285P and VPS41R662 * are recruited to late endosomes / lysosomes . Scale bars 10µm ; zoom of squared area , 1µm .

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D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.


H . VPS41WT / WT , VPS41WT / R662 * and VPS41S285P / R662 * primary fibroblasts incubated with Dextran - 568 for 0 . 5 , 2 , 5 , 8 and 24 hours . Colocalization of Dextran - 568 and SiR - Lysosome Cathepsin D ( SiR - Lyso ) reveals a delay in lysosomal delivery in maternal ( VPS41WT / R662 * ) and patient ( VPS41S285P / R662 * ) cells at 2 hours of Dextran uptake . After 5 hours , maternal cells show similar to control colocalization levels . Patient cells show only after 24 hours colocalization levels similar to control , indicating a delay rather than a block in late endosome - lysosome fusion . > 10 Cells per cell line were quantified .

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H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.


A . Western blot of LC3 expression levels in control and patient fibroblasts . In steady state ( 0 hours ) conditions , patient fibroblasts have a higher ratio of lipidated LC3 ( LC3II ) : LC3I than control cells . Induction of autophagy by nutrient starvation ( 2 hours incubation with minimal EBSS medium ) results in a raise in LC3II : LC3I ratio in both control and patient cells , but in VPS41S285P / R662 * fibroblasts this increase is only modest ( quantified in A ' ) ( n = 2 ) .

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A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).


E . Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP / RFP tandem construct . The increased percentage of GFP / RFP - positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux ( quantified in E ' ) . > 12 Cells per cell line were quantified . Scale bars 10µm ; zoom , 1µm .

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E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.


A . Immunofluorescence of control , parental and patient fibroblasts labeled for LAMP - 1 and mTOR . In steady state conditions ( 0 hours ) VPS41S285P / R662 * fibroblasts show less colocalization between mTOR and LAMP - 1 . VPS41WT / WT , VPS41WT / S285P and VPS41WT / R662 * show an appropriate mTOR response upon nutrient deprivation ( 2 hours ) or restimulation ( 2 hours starvation followed by 15 minutes restimulation ) ( Appendix Fig S5A ) . > 10 Cells per cell line were quantified ( A ' ) . Quantifications are performed relative to colocalization under steady state conditions per cell line ( n = 3 ) . Scale bars , 10µm ; zoom , 1µm .

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A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.


D . Rescue experiments of HeLaVPS41KO cells . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on cellular or secreted secretogranin II ( SgII ) of PC12 cells VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . Cells were washed and incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P .

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B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( strain UA44 ) animals with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( strain UA391 ) fails to rescue neurodegeneration . At day 10 , none of the heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for the α - synuclein ( α - syn ) strain ( total of 90 worms ) and n = 90 for each independent transgenic strain ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative images of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows . Scale bar 20µm .

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C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


A Gene targeting strategy . Left : max genomic locus , targeting construct and max targeted locus . In vitro Flipase ( Flip ) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 and MaxF10 . Right : genomic PCR analysis of tail DNA from F1 mice . PCR products of max wt ( 430 bp ) and max flox ( 526 bp ) alleles . M , 1kb ladder .

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A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) and spleen . Single - cell suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B cells ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , and mature B cells ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) Single - cell suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - )

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)


Flow cytometry analyses of B cell populations ( GFP + ) in spleen . Single - cell suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . spleen follicular B cells ( B220 + CD23 + CD21int ) , marginal zone B cells ( B220 + CD23 - CD21hi ) and immature ( newly formed / transitional stage 1 ) B cells ( B220 + CD23 - CD21lo ) . To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) and spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) and control HET - mb1 ( n = 4 ) mice . Columns represent mean values and error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments .

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F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.


I Cell cycle analysis by flow cytometry of sorted GFP + pre - and pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) and mature ( B220hiIgM + ) bone marrow B cells from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 and HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU and analyzed 4 h later . Single - cell suspensions were prepared , stained and sorted . Sorted cells were subsequently stained with propidium iodide for DNA content . Experiment representative of at least three independent experiments . Dotted line is shown for EdU MFI comparison .

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I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.


J Percentage of annexin - V + GFP + cells in the BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) and control ( n = 4 ) mice . Pro - B , pre - B , immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment representative of at least three independent experiments .

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J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.