BDNF secretion in cultured hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc

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

BDNF secretion in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis


Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of cell lysates showing total TDP - 43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . The antibody used here detects both mouse and human TDP - 43 proteins . GAPDH immunoblotting was used to control for equal loading . Molecular weights are noted in kDa . Bands corresponding to mouse and human TDP - 43 are indicated . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Western blots of cell lysates showing total TDP-43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. The antibody used here detects both mouse and human TDP-43 proteins. GAPDH immunoblotting was used to control for equal loading. Molecular weights are noted in kDa. Bands corresponding to mouse and human TDP-43 are indicated. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; ***, p<0.001 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of TARDBP ( left ) , total SORT1 ( center ) and exon 17b SORT1 ( right ) mRNAs quantified by Q - PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 ( grey bars ) or disease - associated Val337 ( black bars ) TARDBP alleles . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Expression of TARDBP (left), total SORT1 (center) and exon 17b SORT1 (right) mRNAs quantified by Q-PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 (grey bars) or disease-associated Val337 (black bars) TARDBP alleles. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion in stem - cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles , infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

BDNF secretion in stem-cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles, infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment. Results are presented as average ± SEM (N=3 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


A . Ig sequence diversification in wild type and h3 . 3 . Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning . Blue bars = gene conversions ; green lollipops = point mutations ; green bars = ambiguous mutations ( Sale et al , 2001 ) . B . Quantification of sequence diversification in wild type and two h3 . 3 clones expressed as unique events ( gene conversions or point mutations ) per kb per generation . ( Sequences analysed : WT n = 142 , for h3 . 3 c20 n = 140 , for h3 . 3 c32 n = 190 ) .

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

A. Ig sequence diversification in wild type and h3.3. Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning. Blue bars = gene conversions; green lollipops = point mutations; green bars = ambiguous mutations (Sale et al, 2001).B. Quantification of sequence diversification in wild type and two h3.3 clones expressed as unique events (gene conversions or point mutations) per kb per generation. (Sequences analysed: WT n = 142, for h3.3 c20 n = 140, for h3.3 c32 n = 190).


C . Quantification of endogenous AID mRNA in wild type and h3 . 3 cells . The graph represents the mean and SD of four independent RNA extractions of wild type and h3 . 3c20 . p value calculated with the unpaired T test .

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C. Quantification of endogenous AID mRNA in wild type and h3.3 cells. The graph represents the mean and SD of four independent RNA extractions of wild type and h3.3c20. p value calculated with the unpaired T test.


D . Fluctuation analysis for the formation of sIg - loss variants in wild type ( black circles ) and h3 . 3 clones ( black diamonds ) and in two wild type clones ( blue circles ) , two h3 . 3 clones ( blue diamonds ) expressing hAIDup ( Wang et al , 2009 ) at 15 - 20 times the level of wild type AID . The final column shows h3 . 3 cells with hAIDup complemented with H3 . 3 ( blue triangles ) . Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry . Error bars represent mean and 1 S . D . P values calculated with the Mann - Whitney test .

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

D. Fluctuation analysis for the formation of sIg-loss variants in wild type (black circles) and h3.3 clones (black diamonds) and in two wild type clones (blue circles), two h3.3 clones (blue diamonds) expressing hAIDup (Wang et al, 2009) at 15 - 20 times the level of wild type AID. The final column shows h3.3 cells with hAIDup complemented with H3.3 (blue triangles). Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry. Error bars represent mean and 1 S.D. P values calculated with the Mann-Whitney test.


E . Ig sequence diversification in wild type , h3 . 3 and complemented h3 . 3 cells overexpressing hAIDup . Key as in ( A ) plus * = deletion or duplication .

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

E. Ig sequence diversification in wild type, h3.3 and complemented h3.3 cells overexpressing hAIDup. Key as in (A) plus * = deletion or duplication.


F . Mutation rate as a function of hAIDup expression in wild type and h3 . 3 . Filled symbols represent clones overexpressing hAIDup , open symbols clones with natural AID expression . Linear regression lines are shown .

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F. Mutation rate as a function of hAIDup expression in wild type and h3.3. Filled symbols represent clones overexpressing hAIDup, open symbols clones with natural AID expression. Linear regression lines are shown.


B . Diversification in wild type and h3 . 3 cells expressing similar levels of hAIDup and UGI . Red lollipops = transitions at C / T ; Black lollipops = other point mutations ; blue bars = gene conversions .

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B. Diversification in wild type and h3.3 cells expressing similar levels of hAIDup and UGI. Red lollipops = transitions at C/T; Black lollipops = other point mutations; blue bars = gene conversions.


C . Mutation rate as a function of hAIDup expression in UGI - expressing wild type and h3 . 3 cells . Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture .

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C. Mutation rate as a function of hAIDup expression in UGI-expressing wild type and h3.3 cells. Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture.


A . Enrichment of H3 . 3 in the rearranged allele of the Ig light chain ( IGVLR ) compared with the unrearranged allele ( IGVLUR ) . Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs ( * * * * = p < 0 . 0001 , unpaired T - test ) .

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

A. Enrichment of H3.3 in the rearranged allele of the Ig light chain (IGVLR) compared with the unrearranged allele (IGVLUR). Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs (**** = p<0.0001, unpaired T-test).


B . Igλ mRNA levels in wild type and h3 . 3 cells determined by qPCR , normalised to EF - 1α expression and to WT . n = 3 ( ns = not significant , unpaired T - test ) .

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B. Igλ mRNA levels in wild type and h3.3 cells determined by qPCR, normalised to EF-1α expression and to WT. n = 3 (ns = not significant, unpaired T-test).


C . Surface Ig ( sIg ) in multiple clones of wild type ( blue ) and h3 . 3 ( red ) cells . Green = unstained control .

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C. Surface Ig (sIg) in multiple clones of wild type (blue) and h3.3 (red) cells. Green = unstained control.


D . ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR . Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3 , each assayed with 3 qPCR repeats ( statistical analysis as in Panel A ) .

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D. ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR. Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3, each assayed with 3 qPCR repeats (statistical analysis as in Panel A).


E . 4sUDRB mapping of transcription kinetics through IGVL . Representative locations of qPCR amplicons ( blue ) are shown relative to the TSS of IGVL ( distance is shown in parenthesis ) . qPCR signals were normalised to the enrichment at time zero ( before release ) . Error bars represent 1SD of qPCR triplicates .

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E. 4sUDRB mapping of transcription kinetics through IGVL. Representative locations of qPCR amplicons (blue) are shown relative to the TSS of IGVL (distance is shown in parenthesis). qPCR signals were normalised to the enrichment at time zero (before release). Error bars represent 1SD of qPCR triplicates.


A . DNA - RNA hybrids in IGVL and IGCL in wild type and h3 . 3 . The DRIP signal is normalised to total input and to wild type IgVλ ( * * * = p < 0 . 001 , ns = not significant , unpaired T - test ) .

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A. DNA-RNA hybrids in IGVL and IGCL in wild type and h3.3. The DRIP signal is normalised to total input and to wild type IgVλ (*** = p<0.001, ns = not significant, unpaired T-test).


B . MNase mapping reveals that loss of H3 . 3 does not affect average Igλ nucleosome positioning . The red bars indicate the qPCR amplicons for the MNase mapping . The qPCR signals are normalised to the first amplicon at the TSS . Error bars represent 1SD of 2 experimental repeats .

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

B. MNase mapping reveals that loss of H3.3 does not affect average Igλ nucleosome positioning. The red bars indicate the qPCR amplicons for the MNase mapping. The qPCR signals are normalised to the first amplicon at the TSS. Error bars represent 1SD of 2 experimental repeats.


C . DNA - RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI ( red , clones 1 and 2 , each assayed with three qPCR repeats ) or with overexpression of RNAseHI ( blue , clones 3 and 4 , each assayed with three qPCR repeats ) ( * * * = p < 0 . 001 , * * = p < 0 . 01 ; unpaired T - test ) . Expression of hAIDup - FLAG and chickenRNAseHI - YFP in these clones is shown in Figure EV6 .

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

C. DNA-RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI (red, clones 1 and 2, each assayed with three qPCR repeats) or with overexpression of RNAseHI (blue, clones 3 and 4, each assayed with three qPCR repeats) (*** = p<0.001, ** = p < 0.01; unpaired T-test). Expression of hAIDup-FLAG and chickenRNAseHI-YFP in these clones is shown in Figure EV6.


D . Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI .

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D. Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI.


A . Location of ssDNA tracts ≥ 8nt in IGVL in wild type ( WT ) and h3 . 3 cells detected by bisulphite sequencing . Data is from three independent experiments , the results from each being shown as black , red and green bars respectively . ssDNA on the coding strand is shown above the marker line ( numbered relative to the TSS at 0 ) and on the template strand below . In the centre , the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown . B . Quantitation of the bisulphite detection of single stranded DNA in wild type , h3 . 3 and h3 . 3 complemented cells , normalised to wild type . p - values calculated with a  test . Sequences analysed : Wild type n = 322 , h3 . 3n = 320 and h3 . 3 + H3 . 3 n = 95 .

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

A. Location of ssDNA tracts ≥ 8nt in IGVL in wild type (WT) and h3.3 cells detected by bisulphite sequencing. Data is from three independent experiments, the results from each being shown as black, red and green bars respectively. ssDNA on the coding strand is shown above the marker line (numbered relative to the TSS at 0) and on the template strand below. In the centre, the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown.B. Quantitation of the bisulphite detection of single stranded DNA in wild type, h3.3 and h3.3 complemented cells, normalised to wild type. p-values calculated with a  test. Sequences analysed: Wild type n = 322, h3.3n = 320 and h3.3 + H3.3 n = 95.


C . Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression . p - value calculated with a chi ‐ square test . Sequences analysed : RNAseH1 + ve n = 94 , RNAseH1 - ve n = 90 .

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C. Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression. p-value calculated with a chi‐square test. Sequences analysed: RNAseH1+ve n = 94, RNAseH1-ve n = 90.


A . Relative levels of OMA1 mRNA in colorectal cancer tissues ( n = 105 ) compared with adjacent normal tissues ( n = 43 ) from colorectal cancer patients were shown ( using the GEO dataset GSE21510 ) . The data are presented as mean ± SEM , and statistical significance was determined by a Mann - Whitney test . * * * p < 0 . 001 .

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A. Relative levels of OMA1 mRNA in colorectal cancer tissues (n=105) compared with adjacent normal tissues (n=43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann-Whitney test. ***p < 0.001.


B . Kaplan - Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537 . " Low " indicates patients with OMA1 mRNA levels less than the median . " High " indicates patients with OMA1 mRNA levels greater than the median . Statistical analysis was performed using log - rank tests , n = 55 , p = 0 . 0180 .

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

B. Kaplan-Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. "Low" indicates patients with OMA1 mRNA levels less than the median. "High" indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, p=0.0180.


C . The lysates of tumors ( T ) and adjacent normal ( N ) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin . The " c , d and e " bands of OPA1 indicate cleaved OPA1 bands . Tubulin was used as a loading control .

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

C. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The "c, d and e" bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control.


D . Control and OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 ( normoxia ) , 24 , or 48 hours . Cell lysates were then assessed by Western blotting with antibodies against OPA1 , OMA1 , or β - Actin . β - Actin was used as a loading control . Representative immunoblots were from n = 3 independent experiments .

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

D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments.


E . OPA1 - null mouse embryonic fibroblasts ( MEFs ) expressing control ( empty vector ) or OPA1 isoform 1 were cultured in normoxia or hypoxia ( 1 % O2 ) for 24 hours . Cell lysates were analyzed by Western blotting with anti - OPA1 or anti - Tubulin antibody . Representative immunoblots were from n = 3 independent experiments .

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

E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments.


F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours , and the cell lysates were assessed by western blot with antibodies against OMA1 , and GAPDH . β - Actin was used as a loading control ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band . G . The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1 - Flag / β - Actin or E324Q - Flag / β - Actin in hypoxia for 0 , 24 or 48 hours ( n = 3 independent experiments ) . The data are presented as mean ± SD .

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

F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD.


B . Body weight changes of WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice during AOM / DSS treatment . Data are presented as mean ± SEM . Statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 .

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B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01.


C . Typical intestine images on day 105 after mice were euthanized . D . Colorectal lengths ( C ) of WT ( n = 11 biological replicates ) and OMA1 - / - ( n = 12 biological replicates ) mice were measured . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * p < 0 . 05 .

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

C. Typical intestine images on day 105 after mice were euthanized. D. Colorectal lengths (C) of WT (n=11 biological replicates) and OMA1-/- (n=12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, *p < 0.05.


E . Representative images of colorectal tumors from WT ( left ) and OMA1 - / - ( right ) mice . F . The number of tumors in WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice was measured . Each dot represents the tumor number of one individual mouse . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * * * p < 0 . 001 .

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

E. Representative images of colorectal tumors from WT (left) and OMA1-/- (right) mice. F. The number of tumors in WT (n=11) and OMA1-/- (n=12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, ***p < 0.001.


G , H . The number of tumors of different sizes ( diameter ) in each mouse ( G ) and the distribution of different tumor sizes ( H ) were shown ( WT n = 11 biological replicates , and OMA1 - / - n = 12 biological replicates ) . Results are shown as mean ± SEM . p - values were calculated by unpaired student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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

G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n=11 biological replicates, and OMA1-/- n=12 biological replicates). Results are shown as mean ± SEM. p-values were calculated by unpaired student's t-test, **p < 0.01, ***p < 0.001.


I . Histological analysis of colorectal tumors of WT and OMA1 - / - mice was shown by hematoxylin and eosin ( H & E ) staining .

I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining.


J . Relative mRNA expression levels of pro - inflammatory genes ( TNFα , IL - 6 , COX - 2 , CCL2 ) in colorectal homogenates of WT and OMA1 _ KO mice treated with AOM / DSS ( n = 4 - 5 per genotype ) . The data represent the mean ± SEM . , n = 3 independent experiments , and statistical significance was determined by a two - tailed student ' s t - test . * p < 0 . 05 , * * p < 0 . 01 .

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J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01.


K . The colorectal tissues of WT and OMA1 - / - mice were stained with Ki67 .

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K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.


A , B . Control or OMA1 KO HCT116 cells expressing control ( empty vector ) or OMA1 - Flag were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( A ) and lactate production ( B ) were measured by using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD of three independent experiments , statistical significance was assessed by a two - way ANOVA , * P < 0 . 05 . Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH ( A ) . The asterisk indicates a nonspecific band .

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

A, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band.


C . Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h . Subsequent injections of glucose , oligomycin ( Oligo ) , and 2 - deoxy - glucose ( 2 - DG ) were performed as indicated . The extracellular acidification rates ( ECAR ) were measured with a Seahorse Extracellular Flux Analyzer XF96 . The data are presented as mean ± SD ( n = 3 independent experiments ) .

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

C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments).


D . Quantitative analysis of basal and maximal glycolytic capacity . Error bars are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - way ANOVA . N . S . , not significant , * * * p < 0 . 001 .

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

D. Quantitative analysis of basal and maximal glycolytic capacity. Error bars are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-way ANOVA. N.S., not significant, ***p < 0.001.


E , F . Lactate production ( E ) and ATP production ( F ) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2 - DG treatment . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001.


G , H . Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( G ) and lactate production ( H ) were measured , and the values were normalized to the protein concentration . Statistical significance was assessed by a two - way ANOVA ; error bars are presented as mean ± SD of three independent experiments ; N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O 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. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.


A . Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia ( 1 % O2 ) for 24 or 48 hours . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , HK2 , LDHA , PKM2 , GPI , OMA1 , or β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.


B . Control or OMA1 KO Hela cells were untreated ( - ) or CoCl2 - treated ( + ) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours . Cell lysates were assessed by Western blotting with antibodies against HIF - 1α or β - Actin . Relative protein levels were further evaluated by densitometry analysis . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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

B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01.


C . Dihydroethidium ( DHE ) staining of optimum cutting temperature ( OCT ) covered tissues from the colons of WT and OMA1 - / - mice ; DAPI was used as counterstain . DHE was shown in red color , while DAPI in blue color ( representative data from three independent experiments ) . D . Quantification of DHE staining . Error bars are presented as mean ± SD ( n = 3 biological replicates ) , statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 , * * * P < 0 . 001 .

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

C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments). D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001.


E . Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours were stained with mitoSOX , and then analyzed by confocal microscopy ( representative data from three independent experiments ) . F . The fluorescence intensity of mitoSOX was further analyzed by ImageJ software . Bars indicate the mean ± SD of three independent experiments by a two - way ANOVA , * * * p < 0 . 001 .

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

E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments). F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001.


G - I . Control or OMA1 _ KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours , and were treated with DMSO , NAC ( 10 mM ) or Rotenone ( 1 μM ) for 12 hours at the same time . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , and Tubulin ( G ) . The glucose uptake ( H ) and lactate production ( I ) were measured using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - way ANOVA . N . S . , not significant , * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O B-GENEPROD O O B-CELL_LINE 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 B-SMALL_MOLECULE 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 O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O 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. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.


A . 293T cells were transiently co - transfected with empty vector and NDUFB5 - MYC , or OMA1 - E324Q - Flag and NDUFB5 - MYC . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - MYC antibodies ( n = 3 independent experiments ) .

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

A. 293T cells were transiently co-transfected with empty vector and NDUFB5-MYC, or OMA1-E324Q-Flag and NDUFB5-MYC. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-MYC antibodies (n = 3 independent experiments).


B . 293T cells were transiently co - transfected with empty vector and NDUFB6 - GFP , or OMA1 - E324Q - Flag and NDUFB6 - GFP . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) .

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

B. 293T cells were transiently co-transfected with empty vector and NDUFB6-GFP, or OMA1-E324Q-Flag and NDUFB6-GFP. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).


C . 293T cells were transiently co - transfected with empty vector and COX4L1 - GFP , or OMA1 - E324Q - Flag and COX4L1 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - GFP antibodies ( n = 3 independent experiments ) .

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

C. 293T cells were transiently co-transfected with empty vector and COX4L1-GFP, or OMA1-E324Q-Flag and COX4L1-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-GFP antibodies (n = 3 independent experiments).


D . 293T cells were transiently co - transfected with empty vector and NDUFA4 - GFP , or OMA1 - E324Q - Flag and NDUFA4 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) .

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

D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).


E . HCT116 cells stably expressing control ( empty vector ) or OMA1 - Flag , and HCT116 cells infected with control or shOMA1 ( OMA1 knockdown ) lentiviral particles and further cultured for 5 days , were then treated with DMSO or 20 μm CCCP for 4 hours . Cell lysates were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , MT - CO2 , OMA1 or Tubulin . The black arrowhead indicated the full - length COX4L1 or NDUFB6 , and the red arrowhead pointed to the cleaved band . The asterisk indicates a nonspecific band .

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

E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.


F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were treated with CHX ( 100 µg / ml ) plus CCCP ( 20 µM ) for 0 , 2 , 4 , 6 hours . The cell lysates were then assessed by western blot with antibodies against NDUFA4 , COX4L1 , NDUFB5 , NDUFB6 , OMA1 or HSPD1 ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band .

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

F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.


G . Control or OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours . Cell lysates were assessed by Western blotting with anti - NDUFB5 , anti - NDUFB6 , anti - COX4L1 , anti - NDUFA4 antibodies , or anti - β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

G. Control or OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0, 24, or 48 hours. Cell lysates were assessed by Western blotting with anti-NDUFB5, anti-NDUFB6, anti-COX4L1, anti-NDUFA4 antibodies, or anti-β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.


A . Mitochondria isolated from tumors ( T ) or adjacent normal ( N ) tissues of WT and OMA1 - / - mice were subjected to blue native PAGE ( BN - PAGE ) , and respiratory chain complexes were analyzed by Western blotting with anti - NDUFB8 ( Complex I ) , anti - SDHA ( Complex II ) , anti - COX4L1 ( Complex IV ) , and anti - ATP5A1 ( Complex V , ATP synthase ) antibodies . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.


B . Digitonin - solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours , and then analyzed by BN - PAGE and immunoblotting using the indicated antibodies against NDUFB8 ( Complex I ) , SDHA ( Complex II ) , NDUFA4 ( Complex IV ) , and ATP5A1 ( Complex V ) . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * p < 0 . 05 .

O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.


C . The lysates of adjacent normal tissues ( N ) or tumors ( T ) from WT and OMA1 - / - mice were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , UQCRC2 , or Tubulin . Relative protein levels were further evaluated using ImageJ software . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * * p < 0 . 001 .

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

C. The lysates of adjacent normal tissues (N) or tumors (T) from WT and OMA1-/- mice were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, UQCRC2, or Tubulin. Relative protein levels were further evaluated using ImageJ software. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, ***p < 0.001.


Representative confocal images of the FtsZ · SlmA · SBS condensates formed in 150 g / L dextran 500 or Ficoll 70 , and absence of condensates in dilute solution . Scale bars : 20 μm , except for images at higher magnification in dextran ( 5 μm , second row ) .

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

Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).


Turbidity of FtsZ · SlmA · SBS in buffer ( n = 3 ) , in 150 g / L dextran 500 ( n = 5 ) or Ficoll 70 ( n = 3 ) and in 50 g / L PEG 8 ( n = 3 ) . Data correspond to the average ± S . D .

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

Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.


Formation of condensates as a function of FtsZ and SlmA · SBS concentration , as measured by turbidity , in working buffer ( 300 mM KCl ) . SlmA concentration was fivefold that of SBS ( except at 0 . 5 μM SBS , where SlmA concentration was 3 μM ) . Data are the average of 2 independent measurements . Errors ( S . D . ) , symmetrical , are depicted as white discs .

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

Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.


Representative confocal images of the FtsZ · SlmA · SBS condensates at the specified salt concentrations . The concentrations of FtsZ , SlmA and SBS were 12 , 5 and 1 µM , respectively . Scale bars : 5 μm . Data information : All measurements in 150 g / L dextran 500 .

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

Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm. Data information: All measurements in 150 g/L dextran 500.


Representative confocal images showing final state after addition of FtsZ - Alexa 488 to FtsZ · SlmA · SBS complexes ( FtsZ labeled with Alexa 647 ) in 150 g / L dextran . Below , images showing the stepwise diffusion of FtsZ - Alexa 488 into the condensates containing FtsZ - Alexa 647 at the indicated times in seconds ( time zero , beginning of visualization for that particular condensate ) and corresponding intensity profiles at selected times in the green channel . The profile in the red channel is shown as a reference and varies slightly among images . Scale bars : 5 μm ( top row ) and 4 μm ( bottom row ) .

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

Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).


Stepwise diffusion of FtsZ - Alexa 488 into FtsZ · SlmA · SBS condensates ( FtsZ labeled with Alexa 647 ) at the indicated times in seconds ( time zero , beginning of visualization for those particular condensates ) in 100 g / L PEG . Scale bars : 4 μm .

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

Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.


Assembly of FtsZ fibers upon GTP addition ( 0 . 5 mM ) to FtsZ · SlmA · SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes ( time zero , GTP addition ) in 150 g / L dextran . Scale bars : 5 μm . Scheme of the dynamic process on the right . The number of condensates decreases upon fibers formation , and they rearrange upon GTP depletion and fibers disassembly .

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

Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.


B - D . Representative confocal images of the microdroplets without ( B ) and with GTP ( C and D ) . The concentrations of FtsZ , SlmA and SBS were 12 , 5 and 1 μM , respectively ( B and C ) or 6 , 3 and 0 . 5 μM respectively ( D ) . 1 mM ( C ) or 2 mM GTP ( D ) . Scale bars : 40 μm except in images on the far right ( 20 μm ) , which are either a magnification of the indicated region in the merged image ( B ) or independent images at higher magnification ( C , D ) .

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

B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).


B , C . Representative confocal images of the microdroplets stabilized by the E . coli lipid mixture containing the biphasic PEG / DNA mixture and the FtsZ · SlmA · SBS complex without and with 2 mM GTP , respectively . Last image in ( B ) focuses in the lipid interface to show the high density of condensates . Scale bars : 20 μm except in images on the far right ( 10 μm ) , which are an independent image at higher magnification ( B ) or a magnification of the indicated region in the merged image ( C ) .

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

B,C. Representative confocal images of the microdroplets stabilized by the E. coli lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).


( A ) A431 WT and SKO cells - / + GFP - fused WT or A212P - seipin were delipidated , treated with OA for 1 h , fixed and stained with LD540 . Maximum intensity projections of deconvolved confocal z - stacks . Dashed lines indicate cell boundaries . ( B ) Analysis of LD areas of cells treated as in ( A ) and imaged with widefield microscopy . Bars : % of LDs / cell in indicated size category , mean ± SEM , n = 57 - 61 cells / genotype , 2 experiments , p * * < 0 . 005 ( unpaired T - test ) .

O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR 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 O O O O O O O O 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) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test).


( C ) SKO + WT - or A212P - seipin - GFP cells were stained for calreticulin . Maximum intensity projections of deconvolved confocal z - stacks .

O O O 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

(C) SKO + WT- or A212P-seipin-GFP cells were stained for calreticulin. Maximum intensity projections of deconvolved confocal z-stacks.


( D ) SKO + WT - or A212P - seipin - GFP cells were grown in standard growth medium , fixed and stained with anti - GFP antibodies . Widefield and STORM images .

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

(D) SKO + WT- or A212P-seipin-GFP cells were grown in standard growth medium, fixed and stained with anti-GFP antibodies. Widefield and STORM images.


( E ) SKO + WT - seipin - GFP cells were delipidated and treated with OA for 1h , fixed and stained with LipidTox Red . Single focal plane of a 3D - SIM z - stack . ( F ) Analysis of ( E ) , bars : % of LDs / cell with the indicated number of WT - seipin - GFP punctae associated , mean ± SEM , n = 12 cells ( 693 LDs ) , 2 experiments , p * * < 0 . 005 ( Tukey multiple comparisons test ) .

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(E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test).


( G ) SKO + WT - or A212P - seipin - GFP cells were delipidated , incubated with OA for 30 min , and immunolabelled for TEM with anti - GFP antibodies . Black arrowheads : immuno - gold , orange arrowheads : ER - LD junctions . See also Appendix Fig S3 . ( H ) Analysis of ( G ) , bars : relative labeling index in different cellular compartments , n = 3456 - 4512 immuno - gold clusters . Distribution of both WT - and A212P - GFP - seipin immunolabel is non - random ( chi square test , * * p < 0 . 005 ) , but there is significant enrichment of WT - but not A212P - seipin - GFP at ER - LD contacts relative to the ER ( binomial test , * * p < 0 . 005 ) . See also Appendix Table S1 and S2 .

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

(G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2.


( I ) A431 WT cells with seipin tagged with sf - GFP at its chromosomal locus and stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated and treated with OA for 1 h , fixed and stained with LipidTox Red . Single confocal plane of an airyscan z - stack .

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

(I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.


( A ) SKO cells stably expressing WT - seipin - GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30 - 75 min . Airyscan video snapshots , upper panel : an example of 2 seipin - GPF punctae , one stably associated with a LD ( blue arrowhead ) and another not LD associated ( orange arrowhead ) . Lower panel : an example of a mobile LD , with a single seipin puncta moving together with it ( blue arrowhead ) . ( B ) Analysis of ( A ) , bars : mean velocity of tracked seipin puncta ± SEM , n = 13 ROIs from 13 cells , 90 puncta , 2 experiments , * * p < 0 . 005 ( unpaired T - test ) .

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

(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test).


( C ) Analysis of LD mobility in WT and SKO cells . Cells were incubated with OA for 45 - 75 min , LDs were visualized with LD540 and cells imaged by widefield microscopy . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / cell ] , ± SEM , n = 149 - 151 cells , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( D ) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD . ( E ) Analysis of the LD mobility from ( C ) and ( D ) , individual LDs plotted for volume and mean velocity .

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

(C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity.


( F ) WT and SKO cells were transfected with ER marker plasmid ( sfGFP - ER - 3 , green ) , delipidated overnight and incubated with OA and LipidTox Deep Red ( magenta ) for 15 - 75 min . Airyscan video snapshots exemplifying different modes of LDER - associated movement , note the different time scales . Panel I and II : WT cell , panel III : SKO cell . ( G ) Analysis of LDER - associated movement from live airyscan microscopy as in ( F ) . Bars : % of LDs / ROI displaying indicated mode of movement , ± SEM , n = 43 - 55 ROIs ( 201 - 467 LDs ) from 4 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( H ) Analysis of LD velocity . Bars : mean velocity of tracked LDs , ± SEM , n = 29 - 42 LDs / group , 2 experiments , * * p < 0 . 005 ( Kruskal - Wallis test , followed by Dunn test ) .

O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O O O O O O O O O O O O O O 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 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 B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O 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

(F) WT and SKO cells were transfected with ER marker plasmid (sfGFP-ER-3, green), delipidated overnight and incubated with OA and LipidTox Deep Red (magenta) for 15-75 min. Airyscan video snapshots exemplifying different modes of LDER-associated movement, note the different time scales. Panel I and II: WT cell, panel III: SKO cell. (G) Analysis of LDER-associated movement from live airyscan microscopy as in (F). Bars: % of LDs/ROI displaying indicated mode of movement, ± SEM, n= 43-55 ROIs (201-467 LDs) from 4 experiments, **p<0.005 (Mann Whitney test). (H) Analysis of LD velocity. Bars: mean velocity of tracked LDs, ± SEM, n=29-42 LDs/group, 2 experiments, **p<0.005 (Kruskal-Wallis test, followed by Dunn test).


( I ) WT and SKO cells stably expressing HSP47 - APEX were delipidated , treated with OA for 1 h and processed for SB - EM . 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB - EM dataset . Insets are representative cross sections from modeled area highlighting the ER - LD association . Red = LD , Yellow = ER , Green = mitochondria . ( J ) Analysis of LDs from ( I ) . Black lines indicate median , boxes third quartiles , n = 98 - 227 LDs . ( K ) Analysis of LDs displaying no ER contacts from ( I ) . Bars : mean % of LDs in ROIs not in proximity to the ER , ± SEM , n = 11 - 16 ROIs / genotype , * * p < 0 . 005 ( unpaired T - test ) .

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

(I) WT and SKO cells stably expressing HSP47-APEX were delipidated, treated with OA for 1 h and processed for SB-EM. 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB-EM dataset. Insets are representative cross sections from modeled area highlighting the ER-LD association. Red= LD, Yellow= ER, Green=mitochondria. (J) Analysis of LDs from (I). Black lines indicate median, boxes third quartiles, n= 98-227 LDs. (K) Analysis of LDs displaying no ER contacts from (I). Bars: mean % of LDs in ROIs not in proximity to the ER, ± SEM, n= 11-16 ROIs/genotype, **p<0.005 (unpaired T-test).


( A ) WT and SKO cells were treated with OA for 1 h and processed for ET . Representative single tomographic slices of ER - LD contacts . Orange arrowheads indicate narrow ER - LD contacts , blue arrowheads indicate widened ER profiles seen in SKOcells . ( B ) 3D - models of LDs and ER - LD contacts based on tomograms in ( A ) . Yellow : ER , brown : LD , red : ER - LD contacts .

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

(A) WT and SKO cells were treated with OA for 1 h and processed for ET. Representative single tomographic slices of ER-LD contacts. Orange arrowheads indicate narrow ER-LD contacts, blue arrowheads indicate widened ER profiles seen in SKOcells. (B) 3D-models of LDs and ER-LD contacts based on tomograms in (A). Yellow: ER, brown: LD, red: ER-LD contacts.


( C ) Exemplary TEM image of a WT cell incubated with OA for 1h . A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured . Yellow lines indicate measured sites . ( D ) ER profile analysis of ( C ) . Bars : average profile width / cell , ± SEM , n = 11 - 9 cells ( 149 - 224 profiles ) , * p < 0 . 05 ( unpaired T - test ) .

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

(C) Exemplary TEM image of a WT cell incubated with OA for 1h. A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured. Yellow lines indicate measured sites. (D) ER profile analysis of (C). Bars: average profile width/cell, ± SEM, n= 11-9 cells (149-224 profiles), *p<0.05 (unpaired T-test).


( A ) WT and SKO cells stably expressing ACSL3 - Cherry were transfected with ADRP - GFP for 24 h , incubated with OA for 1 h , fixed and stained with LD540 . Single confocal planes of deconvolved confocal z - stacks . ( B ) Colocalization analysis of ( A ) , from single confocal sections . Bars : correlation coefficient m1 ± SEM , n = 34 - 35 cells / genotype , 2 experiments , * * p < 0 . 005 ( unpaired T - test ) .

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(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test).


( C ) WT and SKO cells were transfected with ADRP - GFP for 24 h , incubated with OA for 2 h and co - plated with delipidated WT or SKO cells stably expressing ACSL3 - Cherry . After co - plating , cells were incubated for 2 . 5 h in normal growth medium , then fused together using PEG . Cells were fixed and imaged 1 h post - fusion . Single confocal planes .

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(C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes.


( D ) Colocalization analysis of ( C ) . Bars : correlation coefficient m1 ± SEM , n = 31 - 39 cells / treatment , 3 experiments , * * p < 0 . 005 ( Tukey ' s test ) . Similar results were obtained when cells were kept in delipidation medium after co - plating .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization analysis of (C). Bars: correlation coefficient m1 ± SEM, n=31-39 cells/treatment, 3 experiments, **p<0.005 (Tukey's test). Similar results were obtained when cells were kept in delipidation medium after co-plating.


( E ) WT and SKO cells were treated with OA for 1 h , fixed and stained with anti - ACSL3 antibodies and LD540 . Representative sections of peripheral ER areas , single confocal planes of deconvolved z - stacks . ( F ) Analysis of ( E ) , insets illustrate assigned categories of ACSL3LD association . Bars : % of LDs / ROI in the indicated category , mean ± SEM , n = 9 - 10 peripheral ROIs from 9 - 10 cells / treatment ( 616 - 968 LDs ) , 3 experiments , * * p < 0 . 005 ( unpaired T - test ) .

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

(E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test).


( G ) TEM images of anti - ACSL3 - labeled WT and SKO cells treated with OA for 1 h . ( H ) Analysis of ( G ) , n = 66 - 119 LDs from 8 cells / genotype .

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

(G) TEM images of anti-ACSL3-labeled WT and SKO cells treated with OA for 1 h. (H) Analysis of (G), n= 66-119 LDs from 8 cells/genotype.


( A ) WT and SKO cells stably expressing HPos - Cherry were incubated with OA for 4 h and LDs were labelled with LipidTox Green . The HPos - Cherry signal was bleached and signal recovery to LDs monitored using airyscan imaging . Representative images of 10 acquisitions / genotype , 2 experiments . Circle denotes bleached region , arrowheads indicate HPos signal accumulating around the LD . ( B ) Analysis of HPos - Cherry recovery to LDs after photobleaching , performed by confocal microscopy after 2 - 4 h OA loading . Data is normalized to both pre - bleach and post - bleach intensities , ± SEM , n = 22 - 25 bleached LDs / genotype , 2 experiments .

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

(A) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 4 h and LDs were labelled with LipidTox Green. The HPos-Cherry signal was bleached and signal recovery to LDs monitored using airyscan imaging. Representative images of 10 acquisitions/genotype, 2 experiments. Circle denotes bleached region, arrowheads indicate HPos signal accumulating around the LD. (B) Analysis of HPos-Cherry recovery to LDs after photobleaching, performed by confocal microscopy after 2-4 h OA loading. Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22-25 bleached LDs/genotype, 2 experiments.


( C ) WT and SKO cells were delipidated for 72 h , with the last 24 h in the presence of 1 µM BPY - C12 to label cellular lipids . LD biogenesis was induced by incubating cells in the presence of 0 . 1 mM OA and LipidTox Green for 1 - 2 h . BPY - C12 on LDs was bleached and recovery to LDs monitored using airyscan imaging or conventional confocal imaging . ( D ) Airyscan video snapshots of BPY - C12 recovery to LDs as described in ( C ) . Circle denotes bleached ROI . For the last images of both panels brightness of BPY channel has been increased . ( E ) Analysis of BPY - C12 recovery to LDs from photobleaching experiments described in ( C ) , data is normalized to both pre - bleach and post - bleach intensities , ± SEM , n = 22 bleached LDs / genotype from 3 experiments .

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 I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-SUBCELLULAR O O O O O O

(C) WT and SKO cells were delipidated for 72 h, with the last 24 h in the presence of 1 µM BPY-C12 to label cellular lipids. LD biogenesis was induced by incubating cells in the presence of 0.1 mM OA and LipidTox Green for 1-2 h. BPY-C12 on LDs was bleached and recovery to LDs monitored using airyscan imaging or conventional confocal imaging. (D) Airyscan video snapshots of BPY-C12 recovery to LDs as described in (C). Circle denotes bleached ROI. For the last images of both panels brightness of BPY channel has been increased. (E) Analysis of BPY-C12 recovery to LDs from photobleaching experiments described in (C), data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22 bleached LDs/genotype from 3 experiments.


( F ) Delipidated WT and SKO cells were incubated with OA and LipidTox green for 1 h to induce LD formation , followed by 10 min labeling with BPY - C12 . Live cellconfocal images were captured at indicated chase time points in the presence of OA and LipidTox green . ( G ) Colocalization of BPY - C12 and LDs ( Pearson ' s colocalization correlation coefficient ) ± SEM , n = 8 - 26 cells / time point , 2 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - test , between WT and SKO at the same time point ) .

O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O

(F) Delipidated WT and SKO cells were incubated with OA and LipidTox green for 1 h to induce LD formation, followed by 10 min labeling with BPY-C12. Live cellconfocal images were captured at indicated chase time points in the presence of OA and LipidTox green. (G) Colocalization of BPY-C12 and LDs (Pearson's colocalization correlation coefficient) ± SEM, n=8-26 cells/time point, 2 experiments, *p<0.05, **p<0.005 (unpaired T-test, between WT and SKO at the same time point).


( H ) Delipidated WT and SKO cells were pulsed for 10 min with 0 . 1 mM alkyne - OA either directly ( 1 - 2 ) or after pre - incubation with 0 . 1 mM OA for 1 h ( 3 - 4 ) . After alkyne - OA incubation cells were collected ( 1 and 3 ) or further incubated for 20 min in 10 % FBS medium ( 2 ) or 0 . 1 mM OA medium ( 4 ) . Bars : % of alkyne - OA in neutral lipids ( triacylglycerol and cholesterol esters ) , normalized to WT pulse ( 1 ) , ± SEM , n = 6 - 9 samples , 2 - 3 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - test ) .

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

(H) Delipidated WT and SKO cells were pulsed for 10 min with 0.1 mM alkyne-OA either directly (1-2) or after pre-incubation with 0.1 mM OA for 1 h (3-4). After alkyne-OA incubation cells were collected (1 and 3) or further incubated for 20 min in 10% FBS medium (2) or 0.1 mM OA medium (4). Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to WT pulse (1), ± SEM, n=6-9 samples, 2-3 experiments, *p<0.05, **p<0.005 (unpaired T-test).


( A ) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated and treated with OA for 20 h , fixed and stained with LipidTox Red . Single confocal plane of an airyscan z - stack .

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

(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.


( B ) WT and SKO cells were treated with OA for 20 h , fixed and stained with LD540 . Maximum projections of deconvolved confocal z - stacks . Dashed lines indicate cell boundaries . ( C ) Analysis of LD areas of cells treated as in ( A ) and imaged with widefield microscopy . Bars : % of LDs / field of view in indicated size category , mean ± SEM , n = 25 - 30 fields of view ( 100 - 120 ROIs of not clustered LD areas from 100 - 120 cells ) , 2 experiments , p * * < 0 . 005 ( unpaired T - test ) . See also Appendix Figure S5D .

O O O O O O O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR 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 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 O O 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) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D.


( D ) TEM micrographs of WT and SKO cells treated with OA for 20 h .

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(D) TEM micrographs of WT and SKO cells treated with OA for 20 h.


( E ) WT and SKO cells were treated as in ( B ) , LDs were visualized with LD540 and cells imaged live with widefield microscopy . Exemplary trajectories of peripheral LDs tracked for 40 s . ( F ) Analysis of cells treated and imaged as in ( E ) . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / cell ] , ± SEM , n = 187 - 221 cells , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) .

O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O 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 O O O O O O 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) WT and SKO cells were treated as in (B), LDs were visualized with LD540 and cells imaged live with widefield microscopy. Exemplary trajectories of peripheral LDs tracked for 40 s. (F) Analysis of cells treated and imaged as in (E). Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n=187-221 cells, 2 experiments, **p<0.005 (Mann Whitney test).