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

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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 ) h3 . 3 ( red ) . 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 H3K36me3 in IGVLR IGVLUR . Each bar represents mean of 3 ChIPs for H3K4me3 IgG 2 for H3K36me3 , each assayed with 3 qPCR repeats ( statistical 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 TSS of IGVL ( is shown in parenthesis ) . qPCR signals were normalised to 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 . - hybrids in IGVL IGCL in wild type h3 . 3 . DRIP signal is normalised to total input to wild type IgVλ ( * * * = p < 0 . 001 , ns = not significant , unpaired T - ) .

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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 . red bars indicate qPCR amplicons for MNase mapping . qPCR signals are normalised to first amplicon at TSS . Error bars represent 1SD of 2 experimental repeats .

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

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

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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 in wild type with without RNAseHI overexpression . p - value calculated with a chi ‐ square . 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 of OMA1 mRNA in colorectal cancer tissues ( n = 105 ) compared with adjacent normal tissues ( n = 43 ) from colorectal cancer were shown ( using GEO dataset GSE21510 ) . data are presented as mean ± , statistical significance was determined by a Mann - Whitney . * * * 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 compare between with high low of OMA1 in colorectal cancer samples from GEO dataset GSE17537 . " Low " indicates with OMA1 mRNA less than median . " High " indicates with OMA1 mRNA greater than median . Statistical was performed using log - rank , n = 55 , p = 0 . 0180 .

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

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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 OMA1 KO HCT116 were cultured in hypoxia ( 1 % O2 ) for 0 ( normoxia ) , 24 , or 48 hours . 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 .

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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 . lysates were analyzed by Western blotting with anti - OPA1 or anti - Tubulin antibody . Representative immunoblots were from n = 3 independent experiments .

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

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

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

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

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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 of colorectal tumors of WT OMA1 - / - mice was shown by hematoxylin eosin ( H & E ) .

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


J . Relative mRNA expression of pro - inflammatory genes ( TNFα , IL - 6 , COX - 2 , CCL2 ) in colorectal homogenates of WT OMA1 _ KO mice treated with AOM / DSS ( n = 4 - 5 per genotype ) . data represent mean ± . , n = 3 independent experiments , statistical significance was determined by a two - student ' s t - . * 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 . colorectal tissues of WT 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 expressing control ( empty vector ) or OMA1 - Flag were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . glucose uptake ( A ) lactate production ( B ) were measured by using microplate reader , values were normalized to . Error bars indicate mean ± SD of three independent experiments , statistical significance was assessed by a two - way ANOVA , * P < 0 . 05 . were lysed assessed by western blot with antibodies against OMA1 GAPDH ( A ) . asterisk indicates a nonspecific band .

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

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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 of basal 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 .

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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 ) ATP production ( F ) were measured in control or OPA1 KO HCT116 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 .

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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 were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . glucose uptake ( G ) lactate production ( H ) were measured , values were normalized to . 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 .

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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 OMA1 KO HCT116 were cultured in normoxia or in hypoxia ( 1 % O2 ) for 24 or 48 hours . whole lysates were analyzed by Western blotting with antibodies against HIF - 1α , HK2 , LDHA , PKM2 , GPI , OMA1 , or β - Actin . relative were further evaluated by densitometry using ImageJ software were presented as bar graphs in 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 .

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

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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 ) of optimum cutting ( OCT ) covered tissues from colons of WT 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 . of DHE . 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 .

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

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were cultured in normoxia or hypoxia for 12 hours , were treated with DMSO , NAC ( 10 mM ) or Rotenone ( 1 μM ) for 12 hours at same time . whole lysates were analyzed by Western blotting with antibodies against HIF - 1α , Tubulin ( G ) . glucose uptake ( H ) lactate production ( I ) were measured using microplate reader , values were normalized to . Error bars indicate 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transiently co - transfected with empty vector NDUFB5 - MYC , or OMA1 - E324Q - Flag NDUFB5 - MYC . 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were transiently co - transfected with empty vector NDUFB6 - GFP , or OMA1 - E324Q - Flag NDUFB6 - GFP . 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. 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 were transiently co - transfected with empty vector COX4L1 - GFP , or OMA1 - E324Q - Flag COX4L1 - GFP . 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were transiently co - transfected with empty vector NDUFA4 - GFP , or OMA1 - E324Q - Flag NDUFA4 - GFP . 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 OMA1 KO HCT116 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 . lysates were then assessed by western blot with antibodies against NDUFA4 , COX4L1 , NDUFB5 , NDUFB6 , OMA1 or HSPD1 ( representative data from three independent experiments ) . asterisk indicates a nonspecific band .

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

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 were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours . lysates were assessed by Western blotting with anti - NDUFB5 , anti - NDUFB6 , anti - COX4L1 , anti - NDUFA4 antibodies , or anti - β - Actin . relative were further evaluated by densitometry using ImageJ software were presented as bar graphs in 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OMA1 - / - mice were subjected to blue native PAGE ( BN - PAGE ) , respiratory chain complexes were analyzed by Western blotting with anti - NDUFB8 ( I ) , anti - SDHA ( II ) , anti - COX4L1 ( IV ) , anti - ATP5A1 ( V , ATP synthase ) antibodies . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative were further evaluated by densitometry 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OMA1 KO HCT116 cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours , then analyzed by BN - PAGE immunoblotting using indicated antibodies against NDUFB8 ( I ) , SDHA ( II ) , NDUFA4 ( IV ) , ATP5A1 ( V ) . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative were further evaluated by densitometry 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 O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . lysates of adjacent normal tissues ( N ) or tumors ( T ) from WT OMA1 - / - mice were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , UQCRC2 , or Tubulin . Relative were further evaluated using ImageJ software . Error bars indicate 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 B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) in 50 g / L PEG 8 ( n = 3 ) . Data correspond to average ± S . D .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SlmA · SBS , as measured by turbidity , in working buffer ( 300 mM KCl ) . SlmA was fivefold that of SBS ( except at 0 . 5 μM SBS , where SlmA was 3 μM ) . Data are average of 2 independent measurements . Errors ( S . D . ) , symmetrical , are depicted as discs .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 of FtsZ · SlmA · SBS condensates at specified salt concentrations . concentrations of FtsZ , SlmA SBS were 12 , 5 1 µM , respectively . Scale bars : 5 μm . Data information : measurements in 150 g / L dextran 500 .

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

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 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 , showing stepwise diffusion of FtsZ - Alexa 488 into condensates containing FtsZ - Alexa 647 at indicated times in seconds ( time zero , beginning of visualization for that particular condensate ) corresponding profiles at selected times in green channel . profile in red channel is shown as a reference varies slightly among . Scale bars : 5 μm ( top row ) 4 μm ( bottom row ) .

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

Representative 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 indicated times in seconds ( time zero , beginning of visualization for those particular condensates ) in 100 g / L PEG . Scale bars : 4 μm .

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

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

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

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

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

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

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were stained for calreticulin . Maximum projections of deconvolved confocal z - stacks .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR 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 were grown in standard growth medium , fixed stained with anti - GFP antibodies . Widefield STORM .

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

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

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

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SKO were treated with OA for 1 h 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 ER - LD contacts on tomograms in ( A ) . Yellow : ER , brown : LD , red : ER - LD contacts .

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

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

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 SKO were transfected with ADRP - GFP for 24 h , incubated with OA for 2 h co - plated with delipidated WT or SKO stably expressing ACSL3 - Cherry . After co - plating , were incubated for 2 . 5 h in normal growth medium , then fused together using PEG . were fixed 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 of ( C ) . Bars : correlation coefficient m1 ± , n = 31 - 39 / treatment , 3 experiments , * * p < 0 . 005 ( Tukey ' s ) . Similar results were obtained when 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

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

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

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(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 SKO stably expressing HPos - Cherry were incubated with OA for 4 h LDs were labelled with LipidTox Green . HPos - Cherry signal was bleached signal recovery to LDs monitored using airyscan imaging . Representative of 10 acquisitions / genotype , 2 experiments . Circle denotes bleached region , arrowheads indicate HPos signal accumulating around LD . ( B ) 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 post - bleach intensities , ± , n = 22 - 25 bleached LDs / genotype , 2 experiments .

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

(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 SKO were delipidated for 72 h , with last 24 h in presence of 1 µM BPY - C12 to label lipids . LD biogenesis was induced by incubating in presence of 0 . 1 mM OA LipidTox Green for 1 - 2 h . BPY - C12 on LDs was bleached 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 last of both panels brightness of BPY channel has been increased . ( E ) of BPY - C12 recovery to LDs from photobleaching experiments described in ( C ) , data is normalized to both pre - bleach post - bleach intensities , ± , 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 O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SKO were incubated with OA LipidTox green for 1 h to induce LD formation , followed by 10 min labeling with BPY - C12 . Live cellconfocal were captured at indicated chase time points in presence of OA LipidTox green . ( G ) Colocalization of BPY - C12 LDs ( Pearson ' s colocalization correlation coefficient ) ± , n = 8 - 26 / time point , 2 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - , between WT SKO at same time point ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SKO 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 were collected ( 1 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 cholesterol esters ) , normalized to WT ( 1 ) , ± , n = 6 - 9 samples , 2 - 3 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - ) .

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

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

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

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(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 ) of WT SKO treated with OA for 20 h .

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


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

(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).


( G ) WT SKO were treated with OA for 20 h , fixed stained with anti - ACSL3 antibodies LD540 . Maximum projections of deconvolved confocal z - stacks . Insets show small large LDs . ( H ) of ( G ) . Bars : % of small ( diameter < 0 . 5 µm ) or large ( diameter > 0 . 5 µm ) LDs / in indicated category , mean ± , n = 10 ROIs from 10 / treatment ( 305 - 581 LDs ) , * * p < 0 . 005 ( unpaired T - ) .

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(G) WT and SKO cells were treated with OA for 20 h, fixed and stained with anti-ACSL3 antibodies and LD540. Maximum intensity projections of deconvolved confocal z-stacks. Insets show small and large LDs. (H) Analysis of (G). Bars: % of small (diameter <0.5 µm) or large (diameter >0.5 µm) LDs/cell in the indicated category, mean ± SEM, n=10 ROIs from 10 cells/treatment (305-581 LDs), **p<0.005 (unpaired T-test).


( I ) WT SKO stably expressing HPos - Cherry were incubated with OA for 20 h , LDs were labelled with LipidTox Green prior to imaging . HPos - Cherry signal was bleached signal recovery to LDs was monitored . ( J ) of ( I ) . Data is normalized to both pre - bleach post - bleach intensities , ± , n = 51 - 61 bleached LD areas / genotype , 3 experiments .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 HPos-Cherry were incubated with OA for 20 h, and LDs were labelled with LipidTox Green prior to imaging. The HPos-Cherry signal was bleached and signal recovery to LDs was monitored. (J) Analysis of (I). Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=51-61 bleached LD areas/genotype, 3 experiments.


( A ) Two control BSCL2fibroblast lines were delipidated treated with OA for 1 h , fixed stained with LD540 . Maximum projections of deconvolved confocal z - stacks .

O O O O O O 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) Two control and BSCL2fibroblast cell lines were delipidated and treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks.


( B ) Control BSCL2fibroblasts were incubated with OA for 75 - 90 min , LDs were stained with LD540 imaged live with widefield microscopy . Insets show overlay of two subsequent frames ( 400 ms apart ) . ( C ) of ( B ) . Bars = 1 - [ Pearson ' s colocalization coefficient between subsequent frames / ROI ] , ± , n = 120 - 140 ROIs ( 60 - 70 ) , 2 experiments , * * p < 0 . 005 ( Mann Whitney ) .

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(B) Control and BSCL2fibroblasts were incubated with OA for 75-90 min, LDs were stained with LD540 and cells imaged live with widefield microscopy. Insets show overlay of two subsequent frames (400 ms apart). (C) Analysis of (B). Bars = 1 - [Pearson's colocalization coefficient between subsequent frames/ROI], ± SEM, n=120-140 ROIs (60-70 cells), 2 experiments, **p<0.005 (Mann Whitney test).


( D ) Control BSCL2fibroblasts were transfected with ER marker plasmid ( sfGFP - ER - 3 ) for 24 h treated with OA for 10 - 60 min . LipidTox Deep Red or LD540 was used to label LDs were imaged live by airyscan microscopy . Examples of LDs with high mobility are shown , blue arrowheads indicate a LD moving along ER , orange arrowheads indicate LDs moving independent of ER . ( E ) of ER associated movement of LDs from live airyscan microscopy as in ( D ) . Bars : % of LDs / ROI displaying indicated mode of movement , mean ± , n = 50 - 42 ROIs ( 218 - 374 LDs ) , 2 experiments , * * p < 0 . 005 ( Mann Whitney ) .

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(D) Control and BSCL2fibroblasts were transfected with ER marker plasmid (sfGFP-ER-3) for 24 h and treated with OA for 10-60 min. LipidTox Deep Red or LD540 was used to label LDs and cells were imaged live by airyscan microscopy. Examples of LDs with high mobility are shown, blue arrowheads indicate a LD moving along the ER, orange arrowheads indicate LDs moving independent of the ER. (E) Analysis of ER associated movement of LDs from live airyscan microscopy as in (D). Bars: % of LDs/ROI displaying indicated mode of movement, mean ± SEM, n= 50-42 ROIs (218-374 LDs), 2 experiments, **p<0.005 (Mann Whitney test).


( F ) Control BSCL2fibroblasts were treated with OA for 1 h , fixed stained with anti - ACSL3 antibodies LD540 . Representative sections of peripheral ER areas , maximum projections of deconvolved confocal z - stacks . ( G ) of ( F ) , with insets illustrating assigned categories of ACSL3 LD association . Insets are same as in Fig 4F . Bars : % of LDs / ROI showing category indicated , mean ± , n = 11 - 10 peripheral ROIs from 11 - 10 / group ( 1200 - 2019 LDs ) , 2 experiments , * * p < 0 . 005 ( unpaired T - ) .

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

(F) Control and BSCL2fibroblasts were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, maximum intensity projections of deconvolved confocal z-stacks. (G) Analysis of (F), with insets illustrating assigned categories of ACSL3 LD association. Insets are the same as in Fig 4F. Bars: % of LDs/ROI showing the category indicated, mean ± SEM, n= 11-10 peripheral ROIs from 11-10 cells/group (1200-2019 LDs), 2 experiments, **p<0.005 (unpaired T-test).


( H ) Delipidated fibroblasts were pulsed for 20 min with 0 . 1 mM alkyne - OA either directly ( 1 ) or after pre - incubation with 0 . 1 mM OA for 1 h ( 2 ) , chased in presence of 0 . 1 mM OA for 40 min . Bars : % of alkyne - OA in neutral lipids ( triacylglycerol cholesterol esters ) , normalized to control in ( 1 ) , mean ± , n = 11 - 12 samples , 2 experiments , * p < 0 . 05 ( unpaired T - ) . For C , E , G H results are pooled of 2 controls 2 .

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

(H) Delipidated fibroblasts were pulsed for 20 min with 0.1 mM alkyne-OA either directly (1) or after pre-incubation with 0.1 mM OA for 1 h (2), and chased in the presence of 0.1 mM OA for 40 min. Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to control cells in (1), mean ± SEM, n=11-12 samples, 2 experiments, *p<0.05 (unpaired T-test). For C,E,G and H the results are pooled of 2 controls and 2 patients.


A . Representative FISH showing RAB13 distribution in MDA - MB - 231 . Nuclei outlines are shown in blue green respectively . Arrows points to RAB13 concentrated at protrusive regions . Boxed regions are magnified in insets .

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

A. Representative FISH images showing RAB13 RNA distribution in MDA-MB-231 cells. Nuclei and cell outlines are shown in blue and green respectively. Arrows points to RAB13 RNA concentrated at protrusive regions. Boxed regions are magnified in the insets.


B . Representative immunofluorescence of RAB13 in transfected with indicated siRNAs . Reduction of in RAB13 knockdown confirms specificity of signal . Arrows point to perinuclear RAB13 . Calibration bar shows values . C . Ratios of peripheral / perinuclear calculated from as shown in A B . Bars : mean ± s . e . m . . Values within each bar represent number of observed in 3 independent experiments .

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

B. Representative immunofluorescence images of RAB13 protein in cells transfected with the indicated siRNAs. Reduction of intensity in RAB13 knockdown cells confirms the specificity of the signal. Arrows point to perinuclear RAB13 protein. Calibration bar shows intensity values. C. Ratios of peripheral/perinuclear intensity calculated from images as shown in A and B. Bars: mean ± s.e.m..Values within each bar represent number of cells observed in 3 independent experiments.


D . Protrusions ( Ps ) bodies ( CB ) of induced to migrate towards LPA were isolated analyzed to detect indicated ( by Western blot ; left panels ) or RNAs ( by RT - ddPCR ; right panel ) . Ps / CB enrichment ratios from 2 independent experiments are shown . Bars : mean ± s . e . m . . enrichment of pY397 - FAK serves to verify enrichment of protrusions containing newly formed adhesions in Ps fraction .

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

D. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m..The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction.


B . FISH of mouse fibroblasts expressing β - globin coding followed by indicated UTRs . β - globin is shown in yellow . Nuclei outlines are shown in blue . Arrows points to β - globin concentrated at protrusive regions . ∆ 1 ∆ 1 + 2 indicate deletions of regions shown in ( A ) . Scale bars : 10 μm . C . Distribution of β - globin , or of Ddr2 detected in same , quantified by measuring a Peripheral Distribution Index ( PDI ) . N = 35 - 55 observed in 3 independent experiments . Bars : mean ± 95 % CI . * * * * : p < 0 . 0001 by of variance with Dunnett ' s multiple comparisons .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FISH images of mouse fibroblasts expressing the β-globin coding sequence followed by the indicated UTRs. β-globin RNA is shown in yellow. Nuclei and cell outlines are shown in blue. Arrows points to β-globin RNA concentrated at protrusive regions. ∆1 and ∆1+2 indicate deletions of the regions shown in (A). Scale bars: 10 μm. C. Distribution of β-globin RNA, or of Ddr2 RNA detected in the same cells, quantified by measuring a Peripheral Distribution Index (PDI). N=35-55 cells observed in 3 independent experiments. Bars: mean ± 95% CI. ****:p<0.0001 by analysis of variance with Dunnett's multiple comparisons test.


B . FISH corresponding PDI measurements of mouse fibroblast treated with indicated PMOs . Cyb5r3 is an APC - dependent also enriched at protrusions . Arrows : peripheral Rab13 . Arrowheads : perinuclear Rab13 . Boxed regions are magnified in insets . Note that Rab13 becomes perinuclear in treated with PMOs against GA - rich region . Scale bars : 10 μm . ( 4 μm in insets ) . * * * * : p < 0 . 0001 by of variance with Dunnett ' s multiple comparisons . N = 40 - 90 observed in 3 - 6 independent experiments . Bars : mean ± 95 % CI .

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

B. FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs. Cyb5r3 is an APC-dependent RNA also enriched at protrusions. Arrows: peripheral Rab13 RNA. Arrowheads: perinuclear Rab13 RNA. Boxed regions are magnified in the insets. Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region. Scale bars: 10 μm. (4 μm in insets). ****: p<0.0001 by analysis of variance with Dunnett's multiple comparisons test. N=40-90 cells observed in 3-6 independent experiments. Bars: mean ± 95% CI.


C . Protrusion ( Ps ) ( CB ) fractions were isolated from treated with control - PMO or Rab13 - PMO # 2 . indicated RNAs were detected through nanoString to calculate Ps / CB enrichment ratios ( n = 3 ; bars : mean ± s . e . m . ) . Note that only distribution of Rab13 is affected . * * : p = 0 . 01 by two - way ANOVA with Bonferroni ' s multiple comparisons against corresponding control . D . of indicated RNAs were determined using nanoString from control - or Rab13 PMO # 2 - treated . ( n = 4 ; bars : mean ± s . e . m . ) . No significant differences were detected by two - way ANOVA against corresponding controls .

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

C. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control-PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n=3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **: p=0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control. D. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells. (n=4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls.


A . Schematic showing % GA content positions along human RAB13 3 ' UTR targeted by indicated PMOs . Red rectangles indicate location of GA - rich motifs . Graphs present PDI measurements of RAB13 ( upper panel ) or NET1 ( another APC - dependent ; bottom panel ) detected in MDA - MB - 231 treated with indicated PMOs . PDI = 1 indicates a diffuse distribution . p - values : * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 by of variance with Dunnett ' s multiple comparisons . n . s . : non - significant . N = 30 - 73 observed in 3 - 5 independent experiments . Bars : mean ± s . e . m . .

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

A. Schematic showing %GA content and positions along the human RAB13 3'UTR targeted by the indicated PMOs. Red rectangles indicate the location of GA-rich motifs. Graphs present PDI measurements of RAB13 RNA (upper panel) or NET1 RNA (another APC-dependent RNA; bottom panel) detected in MDA-MB-231 cells treated with the indicated PMOs. PDI=1 indicates a diffuse distribution. p-values: **<0.01, ***<0.001, ****<0.0001 by analysis of variance with Dunnett's multiple comparisons test. n.s.: non-significant. N=30-73 cells observed in 3-5 independent experiments. Bars: mean ± s.e.m..


B . Representative FISH of treated with indicated PMOs . Arrows point to peripheral . Arrowheads point to perinuclear . Boxed regions are magnified in insets . Note that RAB13 becomes perinuclear in treated with PMOs against GA - rich region , while NET1 remains localized at protrusions . Scale bars : 10 μm ( 4 μm in insets ) .

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

B. Representative FISH images of cells treated with the indicated PMOs. Arrows point to peripheral RNA. Arrowheads point to perinuclear RNA. Boxed regions are magnified in the insets. Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region, while NET1 remains localized at protrusions. Scale bars: 10 μm (4 μm in insets).


C . RAB13 were measured by quantitative Western blot normalized to total α - Tubulin or GAPDH ( for representative blot , see Figure 5E ) . Relative in RAB13 PMO - treated compared to control are shown . No significant differences were detected by Kruskal - Wallis with Dunn ' s multiple comparisons . N = 3 - 8 . Bars : mean ± SD .

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C. RAB13 protein levels were measured by quantitative Western blot and normalized to total α-Tubulin or GAPDH levels (for representative blot, see Figure 5E). Relative levels in RAB13 PMO-treated compared to control are shown. No significant differences were detected by Kruskal-Wallis test with Dunn's multiple comparisons test. N=3-8. Bars: mean ± SD.


A . Transwell migration of MDA - MB - 231 treated with control PMOs or RAB13 PMOs ( 191 + 230 ) . reaching bottom surface after 4hrs were counted . n = 25 fields of view in each of 6 independent experiments . Bars : mean ± s . e . m . .

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A. Transwell migration of MDA-MB-231 cells treated with control PMOs or RAB13 PMOs (191+230). Cells reaching the bottom surface after 4hrs were counted. n=25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m..


B . expressing Cherry - NLS , treated with indicated PMOs , were tracked every 5min for 10hrs to derive average migration speed . n = 65 . Bars : mean ± s . e . m . .

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B. Cells expressing Cherry-NLS, and treated with the indicated PMOs, were tracked every 5min for 10hrs to derive average migration speed. n=65 cells. Bars: mean ± s.e.m..


C . PMO - treated were induced to invade through a Matrigel plug . in arbitrary units ( a . u . ) was used to quantify relative invasion from n = 4 independent experiments . Bars : mean ± s . e . m . .

O O O O O O O O O O O O O O O O O O O O O O O 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. PMO-treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n=4 independent experiments. Bars: mean ± s.e.m..


D . Lifeact - GFP - expressing were treated with indicated PMOs imaged every minute over 1 hr . Sequential image frames , from a control , highlight edge retraction ( red arrowheads ) or protrusion ( yellow arrowheads ) . Corresponding edge velocity is shown , with negative values indicating retraction positive values indicating extension . Average protrusion retraction speeds were calculated from n = 11 - 13 . Bars : mean ± s . e . m . . See also movies EV2 EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lifeact-GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n=11-13 cells. Bars: mean ± s.e.m..See also movies EV2 and EV3.


E . treated with indicated PMOs or siRNAs were analyzed by western blot ( upper panels ) to detect RAB13 GAPDH . Migration speed was assessed as in ( B ) from n = 55 - 78 ( bottom graph ) . Bars : mean ± s . e . m . .

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E. Cells treated with the indicated PMOs or siRNAs were analyzed by western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n=55-78 cells (bottom graph). Bars: mean ± s.e.m..


F . Protrusion retraction speed of treated with indicated PMOs or siRNAs was assessed as in ( D ) . Graphs show average normalized values from n = 10 - 13 imaged for 1 hr . Bars : mean ± s . e . m . .

F. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs was assessed as in (D). Graphs show average normalized values from n=10-13 cells imaged for 1 hr. Bars: mean ± s.e.m..


G . Migration speed of RAB13 knockdown re - expressing GFP - RAB13 or GFP - RAB13 with a frameshift ( fs ) mutation at beginning of RAB13 coding region . 28 - 52 were analyzed . Bars : mean ± s . e . m . . Similar results were obtained in two additional independent experiments . Re - expressing were identified through GFP with similar , low GFP signal were tracked . are expressing cherry - NLS for accurate tracking .

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

G. Migration speed of RAB13 knockdown cells re-expressing GFP-RAB13 or GFP-RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28-52 cells were analyzed. Bars: mean ± s.e.m.. Similar results were obtained in two additional independent experiments. Re-expressing cells were identified through GFP fluorescence and cells with similar, low GFP signal were tracked. All cells are expressing cherry-NLS for accurate tracking.


A . Widefield of RAB13 immunofluorescence in MDA - MB - 231 treated with control or RAB13 ( 191 + 230 ) PMOs ratios of peripheral / perinuclear . Scale bars : 10 μm . n = 45 - 50 . Bars : mean ± s . e . m . . Similar results were observed in two additional independent experiments .

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

A. Widefield images of RAB13 immunofluorescence in MDA-MB-231 cells treated with control or RAB13 (191+230) PMOs and ratios of peripheral/perinuclear intensity. Scale bars: 10 μm. n=45-50 cells. Bars: mean ± s.e.m.. Similar results were observed in two additional independent experiments.


B . ( projections of confocal slices throughout height ) of expressing GFP - RAB13 with indicated UTRs . Note that in both cases assumes indistinguishable distribution . Scale bars : 10 μm .

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B. Fluorescence images (projections of confocal slices throughout the cell height) of cells expressing GFP-RAB13 with the indicated UTRs. Note that in both cases the protein assumes indistinguishable distribution. Scale bars: 10 μm.


C . Soluble / particulate fractionation of indicated lines followed by Western blot to detect indicated . RhoGDI TfRc serve as soluble particulate markers , respectively . Graph shows quantitation from n = 3 independent experiments . Bars : mean ± s . e . m . .

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C. Soluble/particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Graph shows quantitation from n=3 independent experiments. Bars: mean ± s.e.m..


Active RAB13 ( RAB13 - GTP ) was pulled - down using MICAL - L1 RBD - GST from indicated PMO - treated amount of endogenous RAB13 was measured by quantitative Western relative of active RAB13 are plotted . n = 8 ( D ) , n = 4 ( E ) . Bars : mean ± s . e . m . .

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Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from the indicated PMO-treated cells The amount of endogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..


E . Active RAB13 ( RAB13 - GTP ) was pulled - down using MICAL - L1 RBD - GST from GFP - RAB13 expressing lines amount of endogenous or exogenous RAB13 was measured by quantitative Western relative of active RAB13 are plotted . n = 8 ( D ) , n = 4 ( E ) . Bars : mean ± s . e . m . .

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

E. Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from GFP-RAB13 expressing lines The amount of endogenous or exogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..


A . Immunoprecipitation Western blot to detect bound to GFP - RAB13 expressed from constructs carrying wt or ∆ PMO RAB13 3 ' UTR . GFP - RAB13 ( T22N ) expresses a nucleotide - free mutant which binds tightly to putative GEFs . RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP - RAB13 . B . of RABIF binding to RAB13 from experiments as in ( A ) . n = 6 . Bars : mean ± s . e . m . .

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

A. Immunoprecipitation and Western blot analysis to detect proteins bound to GFP-RAB13 expressed from constructs carrying wt or ∆PMO RAB13 3'UTR. GFP-RAB13 (T22N) expresses a nucleotide-free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP-RAB13. B. Quantification of RABIF binding to RAB13 from experiments as in (A). n=6. Bars: mean ± s.e.m..


C . Active RAB13 pulldown from with CRISPR knockdown of RABIF using indicated sgRNAs . n = 3 . Bars : mean ± s . e . m . .

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

C. Active RAB13 pulldown assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n=3. Bars: mean ± s.e.m..