( 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 ) .

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

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

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


( A ) Two control 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 .

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

(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 ) .

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

(B) Control 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 ) .

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

(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 ) .

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

(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 O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O

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


A . Representative FISH 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 .

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

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


B . Representative immunofluorescence 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 .

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

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

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


B . FISH 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 .

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

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


B . FISH 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 .

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

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


C . Protrusion ( Ps ) 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 O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A . Schematic showing % GA content 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 . .

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

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


B . Representative FISH 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 ) .

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

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


C . RAB13 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 O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A . Transwell migration of MDA - MB - 231 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 . .

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

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


B . 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 . .

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

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 . .

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

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


D . Lifeact - GFP - expressing 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 .

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

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 . .

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

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 . .

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

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

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


A . Widefield 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 .

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

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


B . 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 .

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

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 . .

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

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 . .

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

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

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


A . Immunoprecipitation 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 . .

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

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


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

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

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 .

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

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 .

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

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

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.


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

B. 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.


( A ) Measurement of maximum glycolytic capacity of MOTS - c in dystrophic muscle cells after oligomycin and 2 - DG treatments ( n = 6 ; * p < 0 . 05 ) .

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

(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 ) .

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

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O

(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 .

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

(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 O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(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).


( 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 ) .

O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(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 .

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

(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 .

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

(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 ) .

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

(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 .

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

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

(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.


( 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 .

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

(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 .

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

(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 .

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

(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 .

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

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

(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.


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 ) .

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

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 ) .

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

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 .

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

(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 ) .

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

(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 ) .

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

(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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(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.


( 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 ) .

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

(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 .

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

(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 ) .

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

(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 ) .

O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(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).


( A Measurement of serum indices including liver enzymes ( AST , ALT and GGT ) from 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 ) to reflect liver and kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) .

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

(A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from 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) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).


B ) Measurement of kidney markers ( CREA and UA ) from 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 ) to reflect liver and kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) .

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

B) Measurement of kidney markers (CREA and UA) from 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) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).


( C ) Morphological examination of liver and kidney from wild - type C57BL / 6 ( C57 ) , untreated mdx controls ( mdx ) and treated mdx mice ( scale bar : 100 μm ) .

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

(C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm).


( D Immunohistochemistry of macrophages , T cells and monocytes in quadriceps and diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . The arrowheads point to CD68 + macrophages , CD3 + T cells or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) .

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

(D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


E ) quantification of macrophages , T cells and monocytes in quadriceps and diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . The arrowheads point to CD68 + macrophages , CD3 + T cells or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) .

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

E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


A . Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons ( DIV16 ) expressing GFP - Sec61β . Zooms represent ( 1 ) an axonal structure with presynaptic boutons ( arrowheads ) , and ( 2 ) a dendritic structure . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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

C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


D . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 288 - 541 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


E . Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non - neuronal tissues . Cereb . , cerebellum . Hippoc . , hippocampus . Spin . , spinal .

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

E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.


F . Pull down assay of HEK293T cells co - expressing Myc - VAPA with BioGFP or BioGFP - SCRN1 . G . Pull down assay of HEK293T cells co - expressing Myc - VAPB with BioGFP or BioGFP - SCRN1 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


H . Scaled representation of SCRN1 - associated proteins identified with pull - down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP - SCRN1 from HEK293T cell lysates with adult rat brain extracts . All candidates showed > 10 enrichment of PSM compared to control .

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

H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.


I . Endogenous localization of SCRN1 and Syt in cortical neurons ( DIV18 ) expressing GFP . Zoom represents an axon structure with presynaptic boutons ( arrowheads ) . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).


J . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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

J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


K . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA alone or SCRN1 shRNA with GFP - SCRN1 . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


A . Localization of GFP - SCRN1 and TagRFP - ER in hippocampal neurons ( DIV16 - 18 ) . Scale bars : 10 µm ( bottom panel , full size ) and 5 µm ( top panel ; bottom panel , zoom ) .

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

A. Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16-18). Scale bars: 10 µm (bottom panel, full size) and 5 µm (top panel; bottom panel, zoom).


B . Localization of GFP - SCRN1 and ssRFP - KDEL in COS7 cells . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

B. Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . Hippocampal neurons ( DIV16 ) co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

C. Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).


D . COS7 cells co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

D. COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).


F . COS7 cells co - expressing HA - VAPB with GFP - SCRN2 or GFP - SCRN3 . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).


B . COS7 cells co - expressing GFP - SCRN1 with HA - VAPB - K87D / M89D . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

B. COS7 cells co-expressing GFP-SCRN1 with HA-VAPB-K87D/M89D. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . COS7 cells co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

C. COS7 cells co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).


D . Hippocampal neurons ( DIV16 ) co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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

D. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).


E . Quantifications of SCRN1 recruitment to VAPB - positive structures in COS7 cells ( % ) . Left graph : co - expression of GFP - SCRN1 with HA - VAPB or HA - VAPB - K87D / M89D ( N = 2 - 3 , n = 44 - 46 ) . Right graph : co - expression of HA - VAPB with GFP or GFP - SCRN1 , GFP - SCRN1 - N , GFP - SCRN1 - C , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A ( N = 2 - 3 , n = 41 - 64 ) . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Chi - square test with post - hoc analysis including Bonferroni correction .

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

E. Quantifications of SCRN1 recruitment to VAPB-positive structures in COS7 cells (%). Left graph: co-expression of GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D ( N=2-3, n= 44-46). Right graph: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A (N=2-3, n=41-64). Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Chi-square test with post-hoc analysis including Bonferroni correction.


F . Pull down of HEK293T cells co - expressing Myc - VAPB with GFP , GFP - SCRN1 , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A .

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

F. Pull down of HEK293T cells co-expressing Myc-VAPB with GFP, GFP-SCRN1, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A.


B . Live hippocampal neurons ( DIV17 - 18 ) co - expressing TagRFP - ER with GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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

B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom).


C . Time - lapse of TagRFP - ER dynamics in hippocampal neurons ( DIV16 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Intact ER structures ( dark arrowheads ) , intact and dynamic ER structures ( light arrowheads ) and impaired an non - dynamic ER structures ( arrows ) are indicated . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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

C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).


E . ER nanostructures visualized with GFP - Sec61β in axons of hippocampal neurons ( DIV18 ) immunostained for α - Tubulin and co - expressed with pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs , and subjected to expansion microscopy . Scale bars : 2 µm ( full size ) and 500 nm ( zoom ) .

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

E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom).


F . FRAP experiment of TagRFP - ER in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . Scale bar : 2 µm .

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

F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm.


G . Average normalized fluorescent TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . H . Normalized fluorescent TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


I . Average normalized fluorescent TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . J . Normalized fluorescent TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


A . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . B . Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

A. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom). B. Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


C . Representative time - lapse of cytosolic GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressed with RIM1a - mCherry to visualize presynaptic sites ( arrowheads ) . Scale bar : 5 µm .

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

C. Representative time-lapse of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressed with RIM1a-mCherry to visualize presynaptic sites (arrowheads). Scale bar: 5 µm.


D . Time - lapses of cytosolic GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressing pSuper control , SCRN1 shRNA or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

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

D. Time-lapses of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressing pSuper control, SCRN1 shRNA or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


E . Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . F . Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

E. Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. F. Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


G . Representative time - lapse of cytosolic GCaMP6f before ( F0 ) and after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressed with mRFP and pSuper empty vector or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

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

G. Representative time-lapse of cytosolic GCaMP6f before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressed with mRFP and pSuper empty vector or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


H . Basal GCaMP6f fluorescence ( F ) normalized to the maximum GCaMP6f fluorescence ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . I . Average basal GCaMP6f fluorescence ( F0 ) normalized to the max GCaMP6f fluorescence intensity ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

H. Basal GCaMP6f fluorescence (F) normalized to the maximum GCaMP6f fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. I. Average basal GCaMP6f fluorescence (F0) normalized to the max GCaMP6f fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


J . Representative time - lapse of cytosolic R - GECO1 before ( F0 ) and after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

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

J. Representative time-lapse of cytosolic R-GECO1 before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


K . Basal R - GECO1 fluorescence ( F ) normalized to the maximum R - GECO1 fluorescence ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . L . Average basal R - GECO1 fluorescence ( F0 ) normalized to the maximum R - GECO1 fluorescence intensity ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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

K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. L. Average basal R-GECO1 fluorescence (F0) normalized to the maximum R-GECO1 fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


B . Flow cytometry for Bu - 1a expression in wild type cells with ( GAA ) n tracts of different length knocked into the BU - 1A locus ( in blue ) . DT40 cells are heterozygous and carry one BU - 1A and one BU - 1B allele . All experiments introducing repeats into BU - 1A are carried out in cells in which the + 3 . 5 G4 has been deleted from both A and B alleles , to avoid transvection between the allele Black outline : positive control ( wild type cells ) ; red outline : negative control ( cells carrying a genetic disruption of BU - 1 )

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

B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)


C , D . Bu - 1a fluctuation analysis of wildtype and primpol cells in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) or with ( GAA ) 10 and ( GAA ) 20 sequence orientated such that it is replicated as the leading ( C ) or lagging ( D ) strand template for a fork entering from the 3 ' end of the locus as shown in panel ( A ) . At least two independent fluctuation analyses were performed . Circles represent the percentage of Bu - 1a loss variants in at least 24 individual clones from these experiments , with mean ± SD reported . * * * * p < 0 . 0001 , * p ≤ 0 . 05 , ns = not significant ; one - way ANOVA .

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

C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.


Human PrimPol , or mutants , tagged with YFP were expressed in primpol cells harbouring ( GAA ) 10 sequence in the BU - 1A locus . Bu - 1a and YFP double positive cells were sorted and expanded for 2 weeks , and then analysed for Bu - 1a expression variants . For each complementation , at least two independently derived clones were subjected to fluctuation analysis . As previously observe expression of hPrimPol [ AxA ] and hPrimPol [ ZfKO ] is deleterious and unstable . Cells expressing these mutations and remaining YFP - positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis . Pooled results from at least three independent fluctuation analyses are represented with mean ± SD indicated with red bar and whiskers . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p ≤ 0 . 05 , ns = not significant ; Kruskal - Wallis

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

Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test


A . DRIP - qPCR analysis reveals accumulation of R - loops across the BU - 1 locus in primpol cells . The DRIP signal was calculated as enrichment over RNase H - treated samples and was normalised to - 0 . 5 kb amplicon . The mean and SD for three biological replicates is presented . An unpaired t - test was used to compare differences between matched amplicons in primpol BU - 1A ( GAA ) 10 and the other cell lines indicated . * * * * p ≤ 0 . 0001 , ns = not

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

A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant