Figure 5A . 293T cells were transiently co - transfected with empty vector and NDUFB5 - MYC , or OMA1 - E324Q - Flag and NDUFB5 - MYC . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - MYC antibodies ( n = 3 independent experiments ) . B . 293T cells were transiently co - transfected with empty vector and NDUFB6 - GFP , or OMA1 - E324Q - Flag and NDUFB6 - GFP . Cell lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) . C . 293T cells were transiently co - transfected with empty vector and COX4L1 - GFP , or OMA1 - E324Q - Flag and COX4L1 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - GFP antibodies ( n = 3 independent experiments ) . D . 293T cells were transiently co - transfected with empty vector and NDUFA4 - GFP , or OMA1 - E324Q - Flag and NDUFA4 - GFP . Cell lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) . E . HCT116 cells stably expressing control ( empty vector ) or OMA1 - Flag , and HCT116 cells infected with control or shOMA1 ( OMA1 knockdown ) lentiviral particles and further cultured for 5 days , were then treated with DMSO or 20 μm CCCP for 4 hours . Cell lysates were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , MT - CO2 , OMA1 or Tubulin . The black arrowhead indicated the full - length COX4L1 or NDUFB6 , and the red arrowhead pointed to the cleaved band . The asterisk indicates a nonspecific band . F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were treated with CHX ( 100 µg / ml ) plus CCCP ( 20 µM ) for 0 , 2 , 4 , 6 hours . The cell lysates were then assessed by western blot with antibodies against NDUFA4 , COX4L1 , NDUFB5 , NDUFB6 , OMA1 or HSPD1 ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band . G . Control or OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours . Cell lysates were assessed by Western blotting with anti - NDUFB5 , anti - NDUFB6 , anti - COX4L1 , anti - NDUFA4 antibodies , or anti - β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 293T cells were transiently co-transfected with empty vector and NDUFB5-MYC, or OMA1-E324Q-Flag and NDUFB5-MYC. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-MYC antibodies (n = 3 independent experiments).B. 293T cells were transiently co-transfected with empty vector and NDUFB6-GFP, or OMA1-E324Q-Flag and NDUFB6-GFP. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).C. 293T cells were transiently co-transfected with empty vector and COX4L1-GFP, or OMA1-E324Q-Flag and COX4L1-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-GFP antibodies (n = 3 independent experiments).D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.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.


Figure 6A . Mitochondria isolated from tumors ( T ) or adjacent normal ( N ) tissues of WT and OMA1 - / - mice were subjected to blue native PAGE ( BN - PAGE ) , and respiratory chain complexes were analyzed by Western blotting with anti - NDUFB8 ( Complex I ) , anti - SDHA ( Complex II ) , anti - COX4L1 ( Complex IV ) , and anti - ATP5A1 ( Complex V , ATP synthase ) antibodies . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B . Digitonin - solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours , and then analyzed by BN - PAGE and immunoblotting using the indicated antibodies against NDUFB8 ( Complex I ) , SDHA ( Complex II ) , NDUFA4 ( Complex IV ) , and ATP5A1 ( Complex V ) . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * p < 0 . 05 . C . The lysates of adjacent normal tissues ( N ) or tumors ( T ) from WT and OMA1 - / - mice were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , UQCRC2 , or Tubulin . Relative protein levels were further evaluated using ImageJ software . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.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.


Figure 1Representative confocal images of the FtsZ · SlmA · SBS condensates formed in 150 g / L dextran 500 or Ficoll 70 , and absence of condensates in dilute solution . Scale bars : 20 μm , except for images at higher magnification in dextran ( 5 μm , second row ) . Turbidity of FtsZ · SlmA · SBS in buffer ( n = 3 ) , in 150 g / L dextran 500 ( n = 5 ) or Ficoll 70 ( n = 3 ) and in 50 g / L PEG 8 ( n = 3 ) . Data correspond to the average ± S . D .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.


Figure 2Formation of condensates as a function of FtsZ and SlmA · SBS concentration , as measured by turbidity , in working buffer ( 300 mM KCl ) . SlmA concentration was fivefold that of SBS ( except at 0 . 5 μM SBS , where SlmA concentration was 3 μM ) . Data are the average of 2 independent measurements . Errors ( S . D . ) , symmetrical , are depicted as white discs . Representative confocal images of the FtsZ · SlmA · SBS condensates at the specified salt concentrations . The concentrations of FtsZ , SlmA and SBS were 12 , 5 and 1 µM , respectively . Scale bars : 5 μm . Data information : All measurements in 150 g / L dextran 500 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm. Data information: All measurements in 150 g/L dextran 500.


Figure 3Representative confocal images showing final state after addition of FtsZ - Alexa 488 to FtsZ · SlmA · SBS complexes ( FtsZ labeled with Alexa 647 ) in 150 g / L dextran . Below , images showing the stepwise diffusion of FtsZ - Alexa 488 into the condensates containing FtsZ - Alexa 647 at the indicated times in seconds ( time zero , beginning of visualization for that particular condensate ) and corresponding intensity profiles at selected times in the green channel . The profile in the red channel is shown as a reference and varies slightly among images . Scale bars : 5 μm ( top row ) and 4 μm ( bottom row ) . Stepwise diffusion of FtsZ - Alexa 488 into FtsZ · SlmA · SBS condensates ( FtsZ labeled with Alexa 647 ) at the indicated times in seconds ( time zero , beginning of visualization for those particular condensates ) in 100 g / L PEG . Scale bars : 4 μm . Assembly of FtsZ fibers upon GTP addition ( 0 . 5 mM ) to FtsZ · SlmA · SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes ( time zero , GTP addition ) in 150 g / L dextran . Scale bars : 5 μm . Scheme of the dynamic process on the right . The number of condensates decreases upon fibers formation , and they rearrange upon GTP depletion and fibers disassembly .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.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.


Figure 4B - 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 ) .

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Figure 4B-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).


Figure 5B , C . Representative confocal images of the microdroplets stabilized by the E . coli lipid mixture containing the biphasic PEG / DNA mixture and the FtsZ · SlmA · SBS complex without and with 2 mM GTP , respectively . Last image in ( B ) focuses in the lipid interface to show the high density of condensates . Scale bars : 20 μm except in images on the far right ( 10 μm ) , which are an independent image at higher magnification ( B ) or a magnification of the indicated region in the merged image ( C ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B,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).


Figure 1 ( A ) A431 WT and SKO cells - / + GFP - fused WT or A212P - seipin were delipidated , treated with OA for 1 h , fixed and stained with LD540 . Maximum intensity projections of deconvolved confocal z - stacks . Dashed lines indicate cell boundaries . ( B ) Analysis of LD areas of cells treated as in ( A ) and imaged with widefield microscopy . Bars : % of LDs / cell in indicated size category , mean ± SEM , n = 57 - 61 cells / genotype , 2 experiments , p * * < 0 . 005 ( unpaired T - test ) . ( C ) SKO + WT - or A212P - seipin - GFP cells were stained for calreticulin . Maximum intensity projections of deconvolved confocal z - stacks . ( D ) SKO + WT - or A212P - seipin - GFP cells were grown in standard growth medium , fixed and stained with anti - GFP antibodies . Widefield and STORM images . ( E ) SKO + WT - seipin - GFP cells were delipidated and treated with OA for 1h , fixed and stained with LipidTox Red . Single focal plane of a 3D - SIM z - stack . ( F ) Analysis of ( E ) , bars : % of LDs / cell with the indicated number of WT - seipin - GFP punctae associated , mean ± SEM , n = 12 cells ( 693 LDs ) , 2 experiments , p * * < 0 . 005 ( Tukey multiple comparisons test ) . ( G ) SKO + WT - or A212P - seipin - GFP cells were delipidated , incubated with OA for 30 min , and immunolabelled for TEM with anti - GFP antibodies . Black arrowheads : immuno - gold , orange arrowheads : ER - LD junctions . See also Appendix Fig S3 . ( H ) Analysis of ( G ) , bars : relative labeling index in different cellular compartments , n = 3456 - 4512 immuno - gold clusters . Distribution of both WT - and A212P - GFP - seipin immunolabel is non - random ( chi square test , * * p < 0 . 005 ) , but there is significant enrichment of WT - but not A212P - seipin - GFP at ER - LD contacts relative to the ER ( binomial test , * * p < 0 . 005 ) . See also Appendix Table S1 and S2 . ( I ) A431 WT cells with seipin tagged with sf - GFP at its chromosomal locus and stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated and treated with OA for 1 h , fixed and stained with LipidTox Red . Single confocal plane of an airyscan z - stack .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test).(C) SKO + WT- or A212P-seipin-GFP cells were stained for calreticulin. Maximum intensity projections of deconvolved confocal z-stacks.(D) SKO + WT- or A212P-seipin-GFP cells were grown in standard growth medium, fixed and stained with anti-GFP antibodies. Widefield and STORM images.(E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test).(G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2.(I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.


Figure 2 ( A ) SKO cells stably expressing WT - seipin - GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30 - 75 min . Airyscan video snapshots , upper panel : an example of 2 seipin - GPF punctae , one stably associated with a LD ( blue arrowhead ) and another not LD associated ( orange arrowhead ) . Lower panel : an example of a mobile LD , with a single seipin puncta moving together with it ( blue arrowhead ) . ( B ) Analysis of ( A ) , bars : mean velocity of tracked seipin puncta ± SEM , n = 13 ROIs from 13 cells , 90 puncta , 2 experiments , * * p < 0 . 005 ( unpaired T - test ) . ( C ) Analysis of LD mobility in WT and SKO cells . Cells were incubated with OA for 45 - 75 min , LDs were visualized with LD540 and cells imaged by widefield microscopy . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / cell ] , ± SEM , n = 149 - 151 cells , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( D ) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD . ( E ) Analysis of the LD mobility from ( C ) and ( D ) , individual LDs plotted for volume and mean velocity . ( F ) WT and SKO cells were transfected with ER marker plasmid ( sfGFP - ER - 3 , green ) , delipidated overnight and incubated with OA and LipidTox Deep Red ( magenta ) for 15 - 75 min . Airyscan video snapshots exemplifying different modes of LDER - associated movement , note the different time scales . Panel I and II : WT cell , panel III : SKO cell . ( G ) Analysis of LDER - associated movement from live airyscan microscopy as in ( F ) . Bars : % of LDs / ROI displaying indicated mode of movement , ± SEM , n = 43 - 55 ROIs ( 201 - 467 LDs ) from 4 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( H ) Analysis of LD velocity . Bars : mean velocity of tracked LDs , ± SEM , n = 29 - 42 LDs / group , 2 experiments , * * p < 0 . 005 ( Kruskal - Wallis test , followed by Dunn test ) . ( I ) WT and SKO cells stably expressing HSP47 - APEX were delipidated , treated with OA for 1 h and processed for SB - EM . 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB - EM dataset . Insets are representative cross sections from modeled area highlighting the ER - LD association . Red = LD , Yellow = ER , Green = mitochondria . ( J ) Analysis of LDs from ( I ) . Black lines indicate median , boxes third quartiles , n = 98 - 227 LDs . ( K ) Analysis of LDs displaying no ER contacts from ( I ) . Bars : mean % of LDs in ROIs not in proximity to the ER , ± SEM , n = 11 - 16 ROIs / genotype , * * p < 0 . 005 ( unpaired T - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test).(C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity.(F) WT and SKO cells were transfected with ER marker plasmid (sfGFP-ER-3, green), delipidated overnight and incubated with OA and LipidTox Deep Red (magenta) for 15-75 min. Airyscan video snapshots exemplifying different modes of LDER-associated movement, note the different time scales. Panel I and II: WT cell, panel III: SKO cell. (G) Analysis of LDER-associated movement from live airyscan microscopy as in (F). Bars: % of LDs/ROI displaying indicated mode of movement, ± SEM, n= 43-55 ROIs (201-467 LDs) from 4 experiments, **p<0.005 (Mann Whitney test). (H) Analysis of LD velocity. Bars: mean velocity of tracked LDs, ± SEM, n=29-42 LDs/group, 2 experiments, **p<0.005 (Kruskal-Wallis test, followed by Dunn test).(I) WT and SKO cells stably expressing HSP47-APEX were delipidated, treated with OA for 1 h and processed for SB-EM. 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB-EM dataset. Insets are representative cross sections from modeled area highlighting the ER-LD association. Red= LD, Yellow= ER, Green=mitochondria. (J) Analysis of LDs from (I). Black lines indicate median, boxes third quartiles, n= 98-227 LDs. (K) Analysis of LDs displaying no ER contacts from (I). Bars: mean % of LDs in ROIs not in proximity to the ER, ± SEM, n= 11-16 ROIs/genotype, **p<0.005 (unpaired T-test).


Figure 3 ( A ) WT and SKO cells were treated with OA for 1 h and processed for ET . Representative single tomographic slices of ER - LD contacts . Orange arrowheads indicate narrow ER - LD contacts , blue arrowheads indicate widened ER profiles seen in SKOcells . ( B ) 3D - models of LDs and ER - LD contacts based on tomograms in ( A ) . Yellow : ER , brown : LD , red : ER - LD contacts . ( C ) Exemplary TEM image of a WT cell incubated with OA for 1h . A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured . Yellow lines indicate measured sites . ( D ) ER profile analysis of ( C ) . Bars : average profile width / cell , ± SEM , n = 11 - 9 cells ( 149 - 224 profiles ) , * p < 0 . 05 ( unpaired T - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) WT and SKO cells were treated with OA for 1 h and processed for ET. Representative single tomographic slices of ER-LD contacts. Orange arrowheads indicate narrow ER-LD contacts, blue arrowheads indicate widened ER profiles seen in SKOcells. (B) 3D-models of LDs and ER-LD contacts based on tomograms in (A). Yellow: ER, brown: LD, red: ER-LD contacts.(C) Exemplary TEM image of a WT cell incubated with OA for 1h. A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured. Yellow lines indicate measured sites. (D) ER profile analysis of (C). Bars: average profile width/cell, ± SEM, n= 11-9 cells (149-224 profiles), *p<0.05 (unpaired T-test).


Figure 4 ( A ) WT and SKO cells stably expressing ACSL3 - Cherry were transfected with ADRP - GFP for 24 h , incubated with OA for 1 h , fixed and stained with LD540 . Single confocal planes of deconvolved confocal z - stacks . ( B ) Colocalization analysis of ( A ) , from single confocal sections . Bars : correlation coefficient m1 ± SEM , n = 34 - 35 cells / genotype , 2 experiments , * * p < 0 . 005 ( unpaired T - test ) . ( C ) WT and SKO cells were transfected with ADRP - GFP for 24 h , incubated with OA for 2 h and co - plated with delipidated WT or SKO cells stably expressing ACSL3 - Cherry . After co - plating , cells were incubated for 2 . 5 h in normal growth medium , then fused together using PEG . Cells were fixed and imaged 1 h post - fusion . Single confocal planes . ( D ) Colocalization analysis of ( C ) . Bars : correlation coefficient m1 ± SEM , n = 31 - 39 cells / treatment , 3 experiments , * * p < 0 . 005 ( Tukey ' s test ) . Similar results were obtained when cells were kept in delipidation medium after co - plating . ( E ) WT and SKO cells were treated with OA for 1 h , fixed and stained with anti - ACSL3 antibodies and LD540 . Representative sections of peripheral ER areas , single confocal planes of deconvolved z - stacks . ( F ) Analysis of ( E ) , insets illustrate assigned categories of ACSL3LD association . Bars : % of LDs / ROI in the indicated category , mean ± SEM , n = 9 - 10 peripheral ROIs from 9 - 10 cells / treatment ( 616 - 968 LDs ) , 3 experiments , * * p < 0 . 005 ( unpaired T - test ) . ( G ) TEM images of anti - ACSL3 - labeled WT and SKO cells treated with OA for 1 h . ( H ) Analysis of ( G ) , n = 66 - 119 LDs from 8 cells / genotype .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test).(C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes.(D) Colocalization analysis of (C). Bars: correlation coefficient m1 ± SEM, n=31-39 cells/treatment, 3 experiments, **p<0.005 (Tukey's test). Similar results were obtained when cells were kept in delipidation medium after co-plating.(E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test).(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.


Figure 5 ( A ) WT and SKO cells stably expressing HPos - Cherry were incubated with OA for 4 h and LDs were labelled with LipidTox Green . The HPos - Cherry signal was bleached and signal recovery to LDs monitored using airyscan imaging . Representative images of 10 acquisitions / genotype , 2 experiments . Circle denotes bleached region , arrowheads indicate HPos signal accumulating around the LD . ( B ) Analysis of HPos - Cherry recovery to LDs after photobleaching , performed by confocal microscopy after 2 - 4 h OA loading . Data is normalized to both pre - bleach and post - bleach intensities , ± SEM , n = 22 - 25 bleached LDs / genotype , 2 experiments . ( C ) WT and SKO cells were delipidated for 72 h , with the last 24 h in the presence of 1 µM BPY - C12 to label cellular lipids . LD biogenesis was induced by incubating cells in the presence of 0 . 1 mM OA and LipidTox Green for 1 - 2 h . BPY - C12 on LDs was bleached and recovery to LDs monitored using airyscan imaging or conventional confocal imaging . ( D ) Airyscan video snapshots of BPY - C12 recovery to LDs as described in ( C ) . Circle denotes bleached ROI . For the last images of both panels brightness of BPY channel has been increased . ( E ) Analysis of BPY - C12 recovery to LDs from photobleaching experiments described in ( C ) , data is normalized to both pre - bleach and post - bleach intensities , ± SEM , n = 22 bleached LDs / genotype from 3 experiments . ( F ) Delipidated WT and SKO cells were incubated with OA and LipidTox green for 1 h to induce LD formation , followed by 10 min labeling with BPY - C12 . Live cellconfocal images were captured at indicated chase time points in the presence of OA and LipidTox green . ( G ) Colocalization of BPY - C12 and LDs ( Pearson ' s colocalization correlation coefficient ) ± SEM , n = 8 - 26 cells / time point , 2 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - test , between WT and SKO at the same time point ) . ( H ) Delipidated WT and SKO cells were pulsed for 10 min with 0 . 1 mM alkyne - OA either directly ( 1 - 2 ) or after pre - incubation with 0 . 1 mM OA for 1 h ( 3 - 4 ) . After alkyne - OA incubation cells were collected ( 1 and 3 ) or further incubated for 20 min in 10 % FBS medium ( 2 ) or 0 . 1 mM OA medium ( 4 ) . Bars : % of alkyne - OA in neutral lipids ( triacylglycerol and cholesterol esters ) , normalized to WT pulse ( 1 ) , ± SEM , n = 6 - 9 samples , 2 - 3 experiments , * p < 0 . 05 , * * p < 0 . 005 ( unpaired T - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 4 h and LDs were labelled with LipidTox Green. The HPos-Cherry signal was bleached and signal recovery to LDs monitored using airyscan imaging. Representative images of 10 acquisitions/genotype, 2 experiments. Circle denotes bleached region, arrowheads indicate HPos signal accumulating around the LD. (B) Analysis of HPos-Cherry recovery to LDs after photobleaching, performed by confocal microscopy after 2-4 h OA loading. Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22-25 bleached LDs/genotype, 2 experiments.(C) WT and SKO cells were delipidated for 72 h, with the last 24 h in the presence of 1 µM BPY-C12 to label cellular lipids. LD biogenesis was induced by incubating cells in the presence of 0.1 mM OA and LipidTox Green for 1-2 h. BPY-C12 on LDs was bleached and recovery to LDs monitored using airyscan imaging or conventional confocal imaging. (D) Airyscan video snapshots of BPY-C12 recovery to LDs as described in (C). Circle denotes bleached ROI. For the last images of both panels brightness of BPY channel has been increased. (E) Analysis of BPY-C12 recovery to LDs from photobleaching experiments described in (C), data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22 bleached LDs/genotype from 3 experiments.(F) Delipidated WT and SKO cells were incubated with OA and LipidTox green for 1 h to induce LD formation, followed by 10 min labeling with BPY-C12. Live cellconfocal images were captured at indicated chase time points in the presence of OA and LipidTox green. (G) Colocalization of BPY-C12 and LDs (Pearson's colocalization correlation coefficient) ± SEM, n=8-26 cells/time point, 2 experiments, *p<0.05, **p<0.005 (unpaired T-test, between WT and SKO at the same time point).(H) Delipidated WT and SKO cells were pulsed for 10 min with 0.1 mM alkyne-OA either directly (1-2) or after pre-incubation with 0.1 mM OA for 1 h (3-4). After alkyne-OA incubation cells were collected (1 and 3) or further incubated for 20 min in 10% FBS medium (2) or 0.1 mM OA medium (4). Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to WT pulse (1), ± SEM, n=6-9 samples, 2-3 experiments, *p<0.05, **p<0.005 (unpaired T-test).


Figure 6 ( A ) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated and treated with OA for 20 h , fixed and stained with LipidTox Red . Single confocal plane of an airyscan z - stack . ( B ) WT and SKO cells were treated with OA for 20 h , fixed and stained with LD540 . Maximum projections of deconvolved confocal z - stacks . Dashed lines indicate cell boundaries . ( C ) Analysis of LD areas of cells treated as in ( A ) and imaged with widefield microscopy . Bars : % of LDs / field of view in indicated size category , mean ± SEM , n = 25 - 30 fields of view ( 100 - 120 ROIs of not clustered LD areas from 100 - 120 cells ) , 2 experiments , p * * < 0 . 005 ( unpaired T - test ) . See also Appendix Figure S5D . ( D ) TEM micrographs of WT and SKO cells treated with OA for 20 h . ( E ) WT and SKO cells were treated as in ( B ) , LDs were visualized with LD540 and cells imaged live with widefield microscopy . Exemplary trajectories of peripheral LDs tracked for 40 s . ( F ) Analysis of cells treated and imaged as in ( E ) . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / cell ] , ± SEM , n = 187 - 221 cells , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.(B) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D.(D) TEM micrographs of WT and SKO cells treated with OA for 20 h.(E) WT and SKO cells were treated as in (B), LDs were visualized with LD540 and cells imaged live with widefield microscopy. Exemplary trajectories of peripheral LDs tracked for 40 s. (F) Analysis of cells treated and imaged as in (E). Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n=187-221 cells, 2 experiments, **p<0.005 (Mann Whitney test).(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 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.


Figure 7 ( 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 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 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 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 ) , 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 .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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 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 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 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), 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.


Figure 1A . 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 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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 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) 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.


Figure 2B . 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 .

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Figure 2B. 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.


Figure 3B . 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. 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) 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.


Figure 4A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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 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 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.


Figure 5A . 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 . 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 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 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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. 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 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 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. 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 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 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.


Figure 6A . 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 . 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 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 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 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 . .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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. 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 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 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 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..


Figure 7A . 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 assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs . n = 3 . Bars : mean ± s . e . m . . D . Quantification of RABIF - RAB13 PLA signal from cells transfected with the indicated siRNAs . Number of observed cells is indicated within each bar . Bars : mean ± s . e . m . . Similar results were observed in one additional independent experiment . E . Representative RABIF - RAB13 PLA images and quantitations from cells transfected with the indicated PMOs . N > 70 cells . Bars : mean ± s . e . m . . Arrows indicate PLA signal at the cell periphery . Arrowheads indicate PLA signal within the cell body . Boxed regions are magnified in the insets . Scale bars : 15 μm ; 5 μm in insets . F . Representative RABIF - GFP PLA images and quantitations from cells expressing GFP - RAB13 ( T22N ) carrying wt or ∆ PMO RAB13 3 ' UTR . N > 70 cells . Bars : mean ± s . e . m . . Arrows indicate PLA signal at the cell periphery . Arrowheads indicate PLA signal within the cell body . Boxed regions are magnified in the insets . Scale bars : 10 μm ; 4 μm in insets .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. 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 assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n=3. Bars: mean ± s.e.m..D. Quantification of RABIF-RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m.. Similar results were observed in one additional independent experiment.E. Representative RABIF-RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets.F. Representative RABIF-GFP PLA images and quantitations from cells expressing GFP-RAB13(T22N) carrying wt or ∆PMO RAB13 3'UTR. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets.


Figure 8B . Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines . Note that even though RABIF binds several - fold more to RAB13 ( T22N ) ( see Figure 7A , B ) it binds similarly to the wild - type and T22N RAB13 RNA , indicating that RABIF binds similarly to nascent RAB13 . After translation , it is likely displaced upon GTP - loading of wild type RAB13 , while it remains more stably bound to the nucleotide - free ( T22N ) form . N = 6 . Bars : mean ± s . e . m . . p - values : * < 0 . 05 by analysis of variance with Dunn ' s multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8B. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several-fold more to RAB13(T22N) (see Figure 7A, B) it binds similarly to the wild-type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP-loading of wild type RAB13, while it remains more stably bound to the nucleotide-free (T22N) form. N=6. Bars: mean ± s.e.m.. p-values: *<0.05 by analysis of variance with Dunn's multiple comparisons test.


Figure 1 ( A ) Measurement of maximum glycolytic capacity of MOTS - c in dystrophic muscle cells after oligomycin and 2 - DG treatments ( n = 6 ; * p < 0 . 05 ) . ( B ) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS - c ( n = 6 ; * p < 0 . 05 ) . ( C ) ATP assay for dystrophic muscle cells in the presence of MOTS - c ( n = 6 ; * p < 0 . 05 ) . NC refers to untreated H2K or H2K mdx cells . ( D ) Tissue distribution and quantitative analysis of Rhodamine B - labeled MOTS - c in mdx mice after single intravenous injection ( 500 μg ) ( n = 6 ; * p < 0 . 05 , * p < 0 . 001 ) . Tissues were harvested and examined 2 h after injection . This experiment was repeated twice . NC refers to untreated mdx mice . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle , D - diaphragm , H - heart , Li - liver , S - spleen , Lu - lung and K - kidney . ( E ) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS - c ( 500 μg ) ( n = 6 ; * p < 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Measurement of maximum glycolytic capacity of MOTS-c in dystrophic muscle cells after oligomycin and 2-DG treatments (n=6; *p<0.05). (B) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS-c (n=6; *p<0.05).  (C) ATP assay for dystrophic muscle cells in the presence of MOTS-c (n=6; *p<0.05). NC refers to untreated H2K or H2K mdx cells.  (D) Tissue distribution and quantitative analysis of Rhodamine B-labeled MOTS-c in mdx mice after single intravenous injection (500 μg) (n=6; *p<0.05, *p<0.001). Tissues were harvested and examined 2 h after injection. This experiment was repeated twice. NC refers to untreated mdx mice. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle, D-diaphragm, H-heart, Li-liver, S-spleen, Lu-lung and K-kidney.  (E) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS-c (500 μg) (n=6; *p<0.05).


Figure 2 ( A ) Measurement of PMO uptake in dystrophic muscle cells ( H2K mdx cells ) in the presence of MOTS - c ( scale bar : 10 μm ) ( n = 6 ; * * p < 0 . 001 ) . PMO - M refers to PMO and MOTS - c ( the same is for the rest unless otherwise specified ) . ( B ) Tissue distribution of FITC - labeled PMO ( 50 mg / kg ) mixed with MOTS - c ( 500 μg ) in mdx mice 48 h after single intravenous injection . NC represents untreated mdx control . The abbreviation is the same as Figure 1D . Color scale represents the fluorescence intensity . ( C ) Quantification of fluorescence intensity in body - wide tissues of mdx mice treated with PMO - M or PMO alone ( n = 3 ; * p < 0 . 05 ) . The comparison was conducted between PMO - M and PMO . ( D , E ) Immunohistochemistry ( D ) and quantitative analysis ( E ) of dystrophin - positive fibers in mdx TA muscles treated with single intramuscular injection of 0 . 5 μg PMO mixed with MOTS - c ( 20 μg ) ( PMO - M ) or PMO alone ( scale bar : 100 μm ) ( n = 3 ; * * p < 0 . 001 ) . C57 means wild - type control C57BL / 6 . ( F ) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO - M or PMO alone ( n = 3 ; * p < 0 . 05 ) . 0 . 5 µg and 2 . 5 µg total protein from C57BL / 6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA muscles from C57BL / 6 were used as normal controls ( the same is for all Western blots unless otherwise specified ) . ( G ) Systemic evaluation of MOTS - c ( 500 μg ) and PMO at the dose of 50 mg / kg / week for 3 weeks in adult mdx mice intravenously . Tissues were examined two weeks after last injection . Immunohistochemistry for dystrophin - positive fibers in body - wide muscles of treated mdx mice ( scale bar : 100 μm ) . C57 means wild - type control C57BL / 6 . ( H , I ) Western blot ( H ) and quantitative analysis ( I ) for dystrophin expression in body - wide muscles from treated mdx mice ( n = 3 ; * p < 0 . 05 ) . 0 . 5 μg , 2 . 5 μg , 5 μg and 10 μg total protein from C57BL / 6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control .

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

Figure 2 (A) Measurement of PMO uptake in dystrophic muscle cells (H2K mdx cells) in the presence of MOTS-c (scale bar: 10 μm) (n=6; **p<0.001). PMO-M refers to PMO and MOTS-c (the same is for the rest unless otherwise specified).  (B) Tissue distribution of FITC-labeled PMO (50 mg/kg) mixed with MOTS-c (500 μg) in mdx mice 48 h after single intravenous injection. NC represents untreated mdx control. The abbreviation is the same as Figure 1D. Color scale represents the fluorescence intensity. (C) Quantification of fluorescence intensity in body-wide tissues of mdx mice treated with PMO-M or PMO alone (n=3; *p<0.05). The comparison was conducted between PMO-M and PMO.  (D, E) Immunohistochemistry (D) and quantitative analysis (E) of dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 0.5 μg PMO mixed with MOTS-c (20 μg) (PMO-M) or PMO alone (scale bar: 100 μm) (n=3; **p<0.001). C57 means wild-type control C57BL/6.  (F) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO-M or PMO alone (n=3; *p<0.05). 0.5 µg and 2.5 µg total protein from C57BL/6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA muscles from C57BL/6 were used as normal controls (the same is for all Western blots unless otherwise specified).  (G) Systemic evaluation of MOTS-c (500 μg) and PMO at the dose of 50 mg/kg/week for 3 weeks in adult mdx mice intravenously. Tissues were examined two weeks after last injection. Immunohistochemistry for dystrophin- positive fibers in body-wide muscles of treated mdx mice (scale bar: 100 μm). C57 means wild-type control C57BL/6.  (H, I) Western blot (H) and quantitative analysis (I) for dystrophin expression in body-wide muscles from treated mdx mice (n=3; *p<0.05). 0.5 μg, 2.5 μg, 5 μg and 10 μg total protein from C57BL/6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control.


Figure 3 ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO - M ( + MOTS - c ) ( scale bar : 100 μm ) . TA refers to tibialis anterior . ( C , D ) Western blot ( C ) and quantitative analysis ( D ) for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO - M ( + MOTS - c ) ( n = 3 ) . 5 µg , 10 µg and 20 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . ( E Immunohistochemistry of dystrophin - positive fibres in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) ( PMO - M ) intravenously ( scale bar : 100 μm ) ( n = 3 ; * p < 0 . 05 ) . PMO represents PMO in saline . F ) quantitative analysis of dystrophin - positive fibres in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) ( PMO - M ) intravenously ( scale bar : 100 μm ) ( n = 3 ; * p < 0 . 05 ) . PMO represents PMO in saline . ( G , H ) Western blot ( G ) and quantitative analysis ( H ) of dystrophin expression in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) intravenously ( n = 3 ; * p < 0 . 05 ) . 0 . 5 µg , 1 µg , 2 . 5 µg , 5 µg and 10 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . Fold change refers to PMO - M relative to PMO alone .

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

Figure 3 (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (scale bar: 100 μm). TA refers to tibialis anterior.  (C, D) Western blot (C) and quantitative analysis (D) for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (n=3). 5 µg, 10 µg and 20 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.  (E Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline.  F) quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline.  (G, H) Western blot (G) and quantitative analysis (H) of dystrophin expression in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) intravenously (n=3; *p<0.05). 0.5 µg, 1 µg, 2.5 µg, 5 µg and 10 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. Fold change refers to PMO-M relative to PMO alone.


Figure 4 Immunohistochemistry of dystrophin - positive fibres in body - wide muscles from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - tailed t test ) . quantitative analysis of dystrophin - positive fibres in body - wide muscles from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - tailed t test ) . ( D ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO alone . 0 . 5 µg , 2 . 5 µg , 5 µg , and 15 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . ( E ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 ; two - tailed t test ) . ( F ) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) , untreated mdx controls ( n = 3 ) and C57BL / 6 ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( G ) Measurement of muscle endurance with the running wheel test for wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . Muscle functional tests were conducted two weeks after last injection .

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

Figure 4 Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).  quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).  (D) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO alone. 0.5 µg, 2.5 µg, 5 µg, and 15 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.  (E) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05; two-tailed t test).  (F) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO-M (n=4) or PMO alone (n=3), untreated mdx controls (n=3) and C57BL/6 (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).  (G) Measurement of muscle endurance with the running wheel test for wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). Muscle functional tests were conducted two weeks after last injection.


Figure 5 ( A ) Measurement of serum creatine kinase ( CK ) levels in wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( B ) Re - localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture ( scale bar : 100 μm ) . The arrowheads point to identical myofibers . C57 means wild - type C57BL / 6 ; mdx represents untreated mdx controls . ( C ) Measurement of body - weight changes of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) . ( D ) Quantitative analysis of centrally nucleated fibres ( CNFs ) in triceps and quadriceps of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( E ) Measurement of cross - sectional area ( CSA ) of muscle fibres from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , two - tailed t test ) .

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

Figure 5 (A) Measurement of serum creatine kinase (CK) levels in wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  (B) Re-localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar: 100 μm). The arrowheads point to identical myofibers. C57 means wild-type C57BL/6; mdx represents untreated mdx controls.  (C) Measurement of body-weight changes of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3).  (D) Quantitative analysis of centrally nucleated fibres (CNFs) in triceps and quadriceps of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman- Keuls test).  (E) Measurement of cross-sectional area (CSA) of muscle fibres from mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, two-tailed t test).