Figure 6 A , B . Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament ( NF ) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR + / - mice shown in ( A ) . Upper panels show total nucleolin stain . Middle panels show merged image of nucleolin ( gray ) , NF and Tuj1 ( magenta ) , and DAPI ( blue ) . Lower panels show nucleolin overlaps with NF and Tuj1 signals as the " axon only " signal . B shows quantification of nucleolin immunofluorescence with approximately a 50 % reduction in nucleolin in the axons of GAR + / - mice in vivo . n = 3 WT , n = 4 GAR + / - ; means ± SEM ; * p < 0 . 05 , unpaired Student ' s t - test . Scale bar - 10 µm . C - F . Representative , exposure matched maximum projection confocal images of FISH for Kpnb1 ( C ) or mTOR ( E ) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT ( left ) or GAR + / - mice . Upper panels for each show total mRNA signal . Middle panels show mRNA ( gray ) signals merged with NF plus Tuj1 ( magenta ) and DAPI ( blue ) . Lower panels show mRNA signal that overlap with NF plus Tuj1 signal ( labeled " axon only " signal ) . Quantification of axonal Kpnb1 ( D ) and mTOR ( F ) mRNA signals compared to the negative control , DapB mRNA , show a significant reduction in these axonal mRNAs in the GAR + / - mice . n = 3 WT , n = 3 GAR + / - ; means ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 unpaired Student ' s t - test . Scale bar - 10 µm .

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

Figure 6 A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm.  C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.


Figure 7 D . Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer , replated and grown for 18h . FISH signal is shown in gray , cell somata ( left ) and axons ( right ) are visualized by neurofilament immunostaining ( magenta ) . Scr - scrambled FISH probe served as a negative control . Scale bar - 10 µm .

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

Figure 7 D. Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer, replated and grown for 18h. FISH signal is shown in gray, cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr - scrambled FISH probe served as a negative control. Scale bar - 10 µm.


Figure 8 A , B . Representative exposure - matched FISH / IF images for DRG neurons co - transfected with control vs . nucleolin ( Ncl ) siRNA plus siRNA - resistant wild type ( WT ; A ) or ∆ GAR ( B ) Ncl cDNAs . Axonal Kpnb1 mRNA is decreased with the Ncl knockdown , and this is not rescued by expression of ∆ GAR Ncl mutant ( scale bars - 10 µm ) .

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

Figure 8 A, B. Representative exposure-matched FISH/IF images for DRG neurons co-transfected with control vs. nucleolin (Ncl) siRNA plus siRNA-resistant wild type (WT; A) or ∆GAR (B) Ncl cDNAs. Axonal Kpnb1 mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant (scale bars - 10 µm).


Figure 9 A . Cultured DRG neurons from wild type ( WT ) Thy1 - YFP mice and Thy1 - YFP / GAR + / - mice ( YFP signal is shown in green ) . Cells were imaged every hour for a period of 48 h . Scale bar - 100 μm . B . Quantification of the time - lapse imaging experiment shown in ( A ) . Growth rate of the longest neurite of each cell was calculated from the time point of starting growth . n = 4 independent biological repeats ; means ± SEM , * * * * p < 0 . 0001 , two - way ANOVA . C . Fluorescence images of cultured DRG neurons from adult C57BL / 6 mice infected with AAV - PHP . s expressing HA - Dendra fused with the wild type nucleolin GAR domain ( GAR WT ) or with the GAR domain with all 10 arginines substituted with asparagines , GAR ( N ) . Neurons were re - plated 9 - 10 days after AAV infection . 24h after re - plating cells were fixed and stained with anti - HA ( grey ) and anti - NFH ( magenta ) antibodies . Scale bar - 100 μm . D . Quantification of total axonal outgrowth and mean process length in HA - positive cells in ( C ) . Outgrowth measurements were based on NFH staining . n = 6 independent biological repeats ; means ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 ( Student ' s - test ) .

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

Figure 9 A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm. B. Quantification of the time-lapse imaging experiment shown in (A). Growth rate of the longest neurite of each cell was calculated from the time point of starting growth. n = 4 independent biological repeats; means ± SEM, **** p < 0.0001, two-way ANOVA.  C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm. D. Quantification of total axonal outgrowth and mean process length in HA-positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; * p < 0.05, *** p < 0.001 (Student's-test).


Figure 1Total number of SNVs / InDels ( allele frequency ≥ 20 % ) detected in primary samples ( x - axis ) plotted against the number of SNVs / InDels detected in corresponding PDX samples ( y - axis ) ; R² coefficient of determination Number of preserved SNVs / InDels ( black bars ) and those that were not detected in PDX models ( grey bars ) in relation to their allele frequencies ( AF ) ; % - fraction of preserved mutationsLarge ( > 1Mb ) CNA ( amplifications - shades of violet ; deletions - shades of red ) identified by low coverage whole genome sequencing in each of the patients and corresponding

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Figure 1Total number of SNVs/InDels (allele frequency≥20%) detected in primary samples (x-axis) plotted against the number of SNVs/InDels detected in corresponding PDX samples (y-axis); R² coefficient of determination Number of preserved SNVs/InDels (black bars) and those that were not detected in PDX models (grey bars) in relation to their allele frequencies (AF); % - fraction of preserved mutationsLarge (>1Mb) CNA (amplifications- shades of violet; deletions - shades of red) identified by low coverage whole genome sequencing in each of the patients and corresponding PDXs


Figure 2Allele frequencies ( AF ) of mutations detected in primary ( PRI ) patients ' samples ( x - axis ) at initial diagnosis ( INI ) and relapse ( REL ) plotted against AF of corresponding mutations detected in matched PDX models ( y - axis ) for relapse type 1 ( left ) and relapse type 2 patients ( right ) ; black - mutations preserved between primary and PDX , blue - mutations , which were selected for / against in PDX models , green - relapse specific mutations selected for in INIPDX , absent or detected with a very low AF in primary patients ' diagnosis sample , orange - subclonal mutations lost in PDX , red - clonal mutations lost in PDX , grey - mutations , which AF was affected by low blast content ( BC ) at relapse Allele frequencies of all mutations preserved between primary patients ' samples ( x - axis ) and PDX samples ( y - axis ) ; PCC - Pearson ' s correlation

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Figure 2Allele frequencies (AF) of mutations detected in primary (PRI) patients' samples (x-axis) at initial diagnosis (INI) and relapse (REL) plotted against AF of corresponding mutations detected in matched PDX models (y-axis) for relapse type 1 (left) and relapse type 2 patients (right); black - mutations preserved between primary and PDX, blue - mutations, which were selected for/against in PDX models, green - relapse specific mutations selected for in INIPDX, absent or detected with a very low AF in primary patients' diagnosis sample, orange - subclonal mutations lost in PDX, red - clonal mutations lost in PDX, grey - mutations, which AF was affected by low blast content (BC) at relapse Allele frequencies of all mutations preserved between primary patients' samples (x-axis) and PDX samples (y-axis); PCC- Pearson's correlation coefficient


Figure 3Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters ( red - low / violet - high methylation levels ) ; * - relapse ( REL ) sample of patient P4 with blast content of 8 % The degree of the mean promoter methylation in patients ' samples plotted against the degree of mean promoter methylation in PDX samples ; ρ - Spearman ' s rank correlation coefficient ; median combined ranks across hexagonal bins are shown as a gradient according to the color

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Figure 3Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters (red - low/violet -high methylation levels); * - relapse (REL) sample of patient P4 with blast content of 8%The degree of the mean promoter methylation in patients' samples plotted against the degree of mean promoter methylation in PDX samples; ρ - Spearman's rank correlation coefficient; median combined ranks across hexagonal bins are shown as a gradient according to the color legend


Figure 4TSS enrichment score of primary samples ( PRI ) and PDXsReads number per peak in primary samples ( x - axis ) plotted against the reads number in corresponding PDX model ; PCC = Pearson ' s correlation coefficient ) Fraction of peaks shared between PDX and corresponding primary leukemia for ATAC peaks with a certain height ( x - axis ; average number of reads that map into an accessible region called by Macs2 ) MA plot of log2 fold changes between primary and PDX samples plotted against the mean of normalized read counts per peak , red - differentially accessible regions ( p < 0 . 05 ) as analyzed by

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Figure 4TSS enrichment score of primary samples (PRI) and PDXsReads number per peak in primary samples (x-axis) plotted against the reads number in corresponding PDX model; PCC=Pearson's correlation coefficient)Fraction of peaks shared between PDX and corresponding primary leukemia for ATAC peaks with a certain height (x-axis; average number of reads that map into an accessible region called by Macs2)MA plot of log2 fold changes between primary and PDX samples plotted against the mean of normalized read counts per peak, red - differentially accessible regions (p<0.05) as analyzed by DESeq


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

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

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


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

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

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


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

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

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


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

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

Figure 4(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test).(C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes.(D) Colocalization analysis of (C). Bars: correlation coefficient m1 ± SEM, n=31-39 cells/treatment, 3 experiments, **p<0.005 (Tukey's test). Similar results were obtained when cells were kept in delipidation medium after co-plating.(E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test).(G) TEM images of anti-ACSL3-labeled WT and SKO cells treated with OA for 1 h. (H) Analysis of (G), n= 66-119 LDs from 8 cells/genotype.


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

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

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


Figure 6 ( A ) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated and treated with OA for 20 h , fixed and stained with LipidTox Red . Single confocal plane of an airyscan z - stack . ( B ) WT and SKO cells were treated with OA for 20 h , fixed and stained with LD540 . Maximum projections of deconvolved confocal z - stacks . Dashed lines indicate cell boundaries . ( C ) Analysis of LD areas of cells treated as in ( A ) and imaged with widefield microscopy . Bars : % of LDs / field of view in indicated size category , mean ± SEM , n = 25 - 30 fields of view ( 100 - 120 ROIs of not clustered LD areas from 100 - 120 cells ) , 2 experiments , p * * < 0 . 005 ( unpaired T - test ) . See also Appendix Figure S5D . ( D ) TEM micrographs of WT and SKO cells treated with OA for 20 h . ( E ) WT and SKO cells were treated as in ( B ) , LDs were visualized with LD540 and cells imaged live with widefield microscopy . Exemplary trajectories of peripheral LDs tracked for 40 s . ( F ) Analysis of cells treated and imaged as in ( E ) . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / cell ] , ± SEM , n = 187 - 221 cells , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( G ) WT and SKO cells were treated with OA for 20 h , fixed and stained with anti - ACSL3 antibodies and LD540 . Maximum intensity projections of deconvolved confocal z - stacks . Insets show small and large LDs . ( H ) Analysis of ( G ) . Bars : % of small ( diameter < 0 . 5 µm ) or large ( diameter > 0 . 5 µm ) LDs / cell in the indicated category , mean ± SEM , n = 10 ROIs from 10 cells / treatment ( 305 - 581 LDs ) , * * p < 0 . 005 ( unpaired T - test ) . ( I ) WT and SKO cells stably expressing HPos - Cherry were incubated with OA for 20 h , and LDs were labelled with LipidTox Green prior to imaging . The HPos - Cherry signal was bleached and signal recovery to LDs was monitored . ( J ) Analysis of ( I ) . Data is normalized to both pre - bleach and post - bleach intensities , ± SEM , n = 51 - 61 bleached LD areas / genotype , 3 experiments .

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

Figure 6(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.(B) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D.(D) TEM micrographs of WT and SKO cells treated with OA for 20 h.(E) WT and SKO cells were treated as in (B), LDs were visualized with LD540 and cells imaged live with widefield microscopy. Exemplary trajectories of peripheral LDs tracked for 40 s. (F) Analysis of cells treated and imaged as in (E). Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n=187-221 cells, 2 experiments, **p<0.005 (Mann Whitney test).(G) WT and SKO cells were treated with OA for 20 h, fixed and stained with anti-ACSL3 antibodies and LD540. Maximum intensity projections of deconvolved confocal z-stacks. Insets show small and large LDs. (H) Analysis of (G). Bars: % of small (diameter <0.5 µm) or large (diameter >0.5 µm) LDs/cell in the indicated category, mean ± SEM, n=10 ROIs from 10 cells/treatment (305-581 LDs), **p<0.005 (unpaired T-test).(I) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 20 h, and LDs were labelled with LipidTox Green prior to imaging. The HPos-Cherry signal was bleached and signal recovery to LDs was monitored. (J) Analysis of (I). Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=51-61 bleached LD areas/genotype, 3 experiments.


Figure 7 ( A ) Two control and BSCL2fibroblast cell lines were delipidated and treated with OA for 1 h , fixed and stained with LD540 . Maximum intensity projections of deconvolved confocal z - stacks . ( B ) Control and BSCL2fibroblasts were incubated with OA for 75 - 90 min , LDs were stained with LD540 and cells imaged live with widefield microscopy . Insets show overlay of two subsequent frames ( 400 ms apart ) . ( C ) Analysis of ( B ) . Bars = 1 - [ Pearson ' s colocalization coefficient between subsequent frames / ROI ] , ± SEM , n = 120 - 140 ROIs ( 60 - 70 cells ) , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( D ) Control and BSCL2fibroblasts were transfected with ER marker plasmid ( sfGFP - ER - 3 ) for 24 h and treated with OA for 10 - 60 min . LipidTox Deep Red or LD540 was used to label LDs and cells were imaged live by airyscan microscopy . Examples of LDs with high mobility are shown , blue arrowheads indicate a LD moving along the ER , orange arrowheads indicate LDs moving independent of the ER . ( E ) Analysis of ER associated movement of LDs from live airyscan microscopy as in ( D ) . Bars : % of LDs / ROI displaying indicated mode of movement , mean ± SEM , n = 50 - 42 ROIs ( 218 - 374 LDs ) , 2 experiments , * * p < 0 . 005 ( Mann Whitney test ) . ( F ) Control and BSCL2fibroblasts were treated with OA for 1 h , fixed and stained with anti - ACSL3 antibodies and LD540 . Representative sections of peripheral ER areas , maximum intensity projections of deconvolved confocal z - stacks . ( G ) Analysis of ( F ) , with insets illustrating assigned categories of ACSL3 LD association . Insets are the same as in Fig 4F . Bars : % of LDs / ROI showing the category indicated , mean ± SEM , n = 11 - 10 peripheral ROIs from 11 - 10 cells / group ( 1200 - 2019 LDs ) , 2 experiments , * * p < 0 . 005 ( unpaired T - test ) . ( H ) Delipidated fibroblasts were pulsed for 20 min with 0 . 1 mM alkyne - OA either directly ( 1 ) or after pre - incubation with 0 . 1 mM OA for 1 h ( 2 ) , and chased in the presence of 0 . 1 mM OA for 40 min . Bars : % of alkyne - OA in neutral lipids ( triacylglycerol and cholesterol esters ) , normalized to control cells in ( 1 ) , mean ± SEM , n = 11 - 12 samples , 2 experiments , * p < 0 . 05 ( unpaired T - test ) . For C , E , G and H the results are pooled of 2 controls and 2 patients .

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

Figure 7(A) Two control and BSCL2fibroblast cell lines were delipidated and treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks.(B) Control and BSCL2fibroblasts were incubated with OA for 75-90 min, LDs were stained with LD540 and cells imaged live with widefield microscopy. Insets show overlay of two subsequent frames (400 ms apart). (C) Analysis of (B). Bars = 1 - [Pearson's colocalization coefficient between subsequent frames/ROI], ± SEM, n=120-140 ROIs (60-70 cells), 2 experiments, **p<0.005 (Mann Whitney test).(D) Control and BSCL2fibroblasts were transfected with ER marker plasmid (sfGFP-ER-3) for 24 h and treated with OA for 10-60 min. LipidTox Deep Red or LD540 was used to label LDs and cells were imaged live by airyscan microscopy. Examples of LDs with high mobility are shown, blue arrowheads indicate a LD moving along the ER, orange arrowheads indicate LDs moving independent of the ER. (E) Analysis of ER associated movement of LDs from live airyscan microscopy as in (D). Bars: % of LDs/ROI displaying indicated mode of movement, mean ± SEM, n= 50-42 ROIs (218-374 LDs), 2 experiments, **p<0.005 (Mann Whitney test).(F) Control and BSCL2fibroblasts were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, maximum intensity projections of deconvolved confocal z-stacks. (G) Analysis of (F), with insets illustrating assigned categories of ACSL3 LD association. Insets are the same as in Fig 4F. Bars: % of LDs/ROI showing the category indicated, mean ± SEM, n= 11-10 peripheral ROIs from 11-10 cells/group (1200-2019 LDs), 2 experiments, **p<0.005 (unpaired T-test).(H) Delipidated fibroblasts were pulsed for 20 min with 0.1 mM alkyne-OA either directly (1) or after pre-incubation with 0.1 mM OA for 1 h (2), and chased in the presence of 0.1 mM OA for 40 min. Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to control cells in (1), mean ± SEM, n=11-12 samples, 2 experiments, *p<0.05 (unpaired T-test). For C,E,G and H the results are pooled of 2 controls and 2 patients.


Figure 1 . ( A ) Western blot analysis for pRb in DM - 2 cultures . ( B ) Immunostaining for MHC in DM - 3 and DM - 6 differentiating myotubes . Nuclei were stained with DAPI ( blue ) . ( C ) Mean number of myotubes on the indicated days after differentiation . Each time point is mean ± SD of six fields from six independent experiments . ( D ) TUNEL staining . Arrowheads indicate TUNEL - positive nuclei ( green ) . ( E ) LC3 - RFP expression in control ( ctrl ) and Rb − / − myotubes . Insets show nuclear DAPI stain ( blue ) . ( F ) Representative Western blot ( n = 3 ) for LC3 . Chloroquine was added 12 h before cell harvest . ( G ) Representative Western blot for LC3 in E16 . 5 control and mgRb : Rb − / − muscles . ( H ) Quantification of LC3 - II / tubulin ratio in E16 . 5 mgRb : Rb − / − skeletal muscles relative to control as depicted in G ( mean ± SD ; n = 3 ) . * , P = 0 . 016 by Student ' s t test . ( I ) Representative Western blot for LC3 - I and LC3 - II expression in whole cell lysates from the indicated myotubes transduced with Ad . GFP or Ad . RbΔK11 . ( J ) Quantification of LC3 - II / tubulin ratio by Western blots as shown in I . Data represent mean ± SD ( n = 7 ) . * , P = 0 . 047 ; * * , P = 0 . 005 by analysis of variance .

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

Figure 1.(A) Western blot analysis for pRb in DM-2 cultures.(B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments.(D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green).(E) LC3-RFP expression in control (ctrl) and Rb−/− myotubes. Insets show nuclear DAPI stain (blue).(F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest.(G) Representative Western blot for LC3 in E16.5 control and mgRb:Rb−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student's t test.(I) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.


Figure 2 . ( A , left ) MitoTracker staining . ( right ) MitoTracker staining after bezafibrate treatment . ( B ) Quantification of myotubes with perinuclear MitoTracker - positive aggregates in untreated ( vehicle ) or bezafibrate - treated myotubes at DM - 2 . Data represent mean ± SD ( n = 3 ) . * , P < 0 . 001 by Student ' s t test . ( C ) MitoTracker ( red ) and LC3immunostaining ( green ) of myotubes at DM - 2 . Chloroquine and 3 - MA ( where indicated ) were added 12 h before staining . Note the extensive overlap of MitoTracker and LC3 in Rb − / − myotubes ( yellow ) , which is inhibited by 3 - MA . ( D ) Mean percentage of MitoTracker ( red ) to LC3 ( green ) overlap in 25 myotubes from two independent cultures in control versus Rb − / − myotubes ( total ) or Rb − / − myotubes with perinuclear mitochondrial aggregation ( high ) . * , P = 7 × 10 − 5 by Student ' s t test . ( E ) Ultrastructural analysis of myotubes treated or not with 3 - MA at DM - 2 . Arrowheads point to mitochondria . Asterisks indicate electron - dense autolysosomes . N , nucleus . ( F ) Ratio of mitochondrialDNA / nuclearDNA in the indicated cultures ( n = 3 ) . ( G ) Ratio of mitochondrialDNA / nuclearDNA in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 009 ) . ( H ) Quantification of ATP in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 011 ) . ( F - H ) Error bars represent SD . * , P < 0 . 001 .

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

Figure 2.(A, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student's t test.(C) MitoTracker (red) and LC3immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/−myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/−myotubes (total) or Rb−/−myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student's t test.(E) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrialDNA/nuclearDNA in the indicated cultures (n = 3). (G) Ratio of mitochondrialDNA/nuclearDNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F-H) Error bars represent SD. *, P < 0.001.


Figure 3 . ( A ) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad . GFP or Ad . Bcl - 2 . Each point represents the mean ± SD of six fields from six independent experiments . Bcl - 2 - rescued , twitching Rb − / − myotubes are shown in Video 2 . ( inset ) Western blot for Bcl - 2 in Ad . Bcl2 - transduced versus uninfected primary myoblasts . ( B ) Immunostaining for MCK ( green ) in Ad . Bcl - 2 - transduced control ( ctrl ) and Rb − / − myotubes at DM - 6 . ( C ) Western blot for MCK , myogenin , MHC , and troponin T in indicated cultures at DM - 6 after transduction with Ad . Bcl - 2 . Tubulin was used as a loading control . Black lines indicate that intervening lanes have been spliced out . ( D ) Mean number of Rb − / − myotubes after 3 - MA treatment , bezafibrate , or DMSO / vehicle as indicated . Counts are mean ± SD of six fields ( n = 4 ) . ( E , left and middle ) Brightfield images of the indicated myotubes treated or not with 3 - MA at DM - 8 . ( right ) Immunostaining for MHC ( red ) at DM - 14 in the presence of 3 - MA . Arrowheads label 3 - MA - rescued Rb − / − myotubes at DM - 8 . 3 - MA - rescued , twitching Rb − / − myotubes are shown in Video 4 . ( F ) Immunostaining for BrdU ( green ) and MHC ( red ) of 3 - MA - treated cultures at DM - 2 or DM - 10 . Rb − / − myotubes incorporated BrdU at DM - 2 ( arrowheads ) but not DM - 10 . ( G ) Brightfield images ( left and middle ) and MHC staining ( right ) of the indicated cultures treated or not with bezafibrate . Arrowheads point to bezafibrate - rescued Rb − / − myotubes .

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

Figure 3.(A) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad.GFP or Ad.Bcl-2. Each point represents the mean ± SD of six fields from six independent experiments. Bcl-2-rescued, twitching Rb−/− myotubes are shown in Video 2. (inset) Western blot for Bcl-2 in Ad.Bcl2-transduced versus uninfected primary myoblasts.(B) Immunostaining for MCK (green) in Ad.Bcl-2-transduced control (ctrl) and Rb−/− myotubes at DM-6.(C) Western blot for MCK, myogenin, MHC, and troponin T in indicated cultures at DM-6 after transduction with Ad.Bcl-2. Tubulin was used as a loading control. Black lines indicate that intervening lanes have been spliced out.(D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4).(E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4.(F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10.(G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.


Figure 4 . ( A ) Immunostaining for MHC ( green ) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine . Note the shortened myotubes in the presence of lonidamine . ( B ) Representative immunoblot ( left ) and mean expression of myogenin and MCK normalized for MHC expression ( right ; n = 3 ) . ( C ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunoblotted for pRb . Tubulin was used as a loading control . ( D ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre and immunostained for pRb ( red ) . ( insets ) DAPI staining for nuclei ( blue ) . ( E ) Immunostaining for MHC in Rbf / f myoblasts transduced with Ad . Cre and induced to differentiate in normoxia ( top and middle ) or hypoxia ( bottom ) . ( F , top ) Brightfield images of Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . ( bottom ) Larger view of the boxed field . Arrowheads point to rescued Rb − / − myotubes . This field is shown in Video 6 . ( G ) Quantification of myotube formation in Rbf / f myoblasts transduced with Ad . EV or Ad . Cre and induced to differentiate under hypoxia . Counts are mean of six representative fields ( n = 3 ) . * , P = 0 . 004 . ( H ) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions . Myotube counts are the mean of six fields ( n = 4 ) . ( I ) MHC staining ( red ) of the indicated myoblasts transduced with Ad . NF - κB / HIF - 1α and induced to differentiate in normoxia . ( J ) Quantification of myotube formation in the indicated myoblasts transduced with Ad . EV or Ad . NF - κB / HIF - 1α and induced to differentiate in normoxia . Myotube counts are the mean of six fields ( n = 3 ) . Error bars represent SD .

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

Figure 4.(A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine.(B) Representative immunoblot (left) and mean expression of myogenin and MCK normalized for MHC expression (right; n = 3).(C) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunoblotted for pRb. Tubulin was used as a loading control.(D) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunostained for pRb (red). (insets) DAPI staining for nuclei (blue).(E) Immunostaining for MHC in Rbf/f myoblasts transduced with Ad.Cre and induced to differentiate in normoxia (top and middle) or hypoxia (bottom).(F, top) Brightfield images of Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. (bottom) Larger view of the boxed field. Arrowheads point to rescued Rb−/− myotubes. This field is shown in Video 6.(G) Quantification of myotube formation in Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. Counts are mean of six representative fields (n = 3). *, P = 0.004.(H) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions. Myotube counts are the mean of six fields (n = 4).(I) MHC staining (red) of the indicated myoblasts transduced with Ad.NF-κB/HIF-1α and induced to differentiate in normoxia.(J) Quantification of myotube formation in the indicated myoblasts transduced with Ad.EV or Ad.NF-κB/HIF-1α and induced to differentiate in normoxia. Myotube counts are the mean of six fields (n = 3). Error bars represent SD.


Figure 1 ( B , C ) Levels of anti - HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL - OVA / CFA following adoptive transfer of BHEL cells without CD4OT - II cells ( no T cell control ) or with control CD4OT - II cells or NEF - expressing CD4OT - II cells . ( B ) NEF WT of HIV - 1SF2 ( C ) patient - derived NEF variants . Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1 . Each dot represents one animal . Bars represent mean values with SD from 2 - 10 mice analyzed from 3 ( B ) and 2 ( C ) independent experiments . Statistical significance was assessed by Mann Whitney U test . * * * P ≤ 0 . 001 , * P ≤ 0 .

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

Figure 1(B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05


Figure 2 ( E ) Representative confocal micrographs of p - tyrosine ( p - Tyr ) polarization at B - T cell contacts after 1 - 2h of co - culture . Cell conjugates were stained with anti - pTyr antibody . Scale bar , 5µm . The top panels display only the p - Tyr signal in grey , the bottom panel depicts p - Tyr ( red ) , BHEL cells ( blue ) and CD4OT - II cells ( green ) . ( F ) Quantification of p - Tyr accumulation at the B - T interface as shown in E . Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment . ( G ) NEF prevents induction of F - actin remodeling in BHEL during IS . BHEL cells ( blue ) and CD4OT - II cells ( green ) were placed on PLL - coated slides and subjected to live - cell time - lapse imaging . Shown are representative still images from the indicated time points post contact formation ( see Movies EV1 and EV2 ) . Red signals indicate actin polymerization specifically in BHEL cells . Scale bars , 5µm . Filled and empty arrowheads indicate examples of accumulation of polarized actin or p - Tyr signal at B - T interface during IS formation or lack thereof , respectively . Asterisks mark pairs of interacting T and B cell . ( H ) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment . ( L ) Relative B cell proliferation ( quantified by gating on the last 3 - 4 generations ) is plotted as mean values with SD from four independent experiments . Control T cells + Ag were arbitrarily set to 1 .

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

Figure 2(E) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment. (G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment. (L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.


Figure 3 ( B ) Production of anti - HEL IgG at the indicated day post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and CD4OT - II cells expressing NEF or various NEF mutants . Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1 . Each dot represents one animal . Shown are mean values with SD from 5 - 12 mice from 4 experiments . ( D ) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co - cultured with control T cells arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition . ( H ) Relative surface expression of CD4 , CD28 , OX40 , ICOS , CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants . Analysis of surface expression was performed using flow cytometry 3 - 7 d post transduction of T cells . Depicted are mean values with SD from 3 - 6 independent experiments , with control T cells arbitrarily set to 100 % as indicated by the dashed line . ( I ) Representative confocal images of the subcellular distribution of IL - 4 in CD4OT - II cells expressing the indicated proteins engaged in an IS with BHEL cells . B cells and T cells were placed on PLL - coated cover glasses for 60 - 90min , fixed and stained for intracellular IL - 4 . Filled and empty arrowheads indicate presence or absence of IL - 4 accumulation at the IS , respectively . Scale bars , 5µm . ( J ) Quantification of IL - 4 accumulation at the IS as depicted in I . Shown are mean values with SD from 3 independent experiments relative to control cells set to 100 % .

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Figure 3(B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments.(D) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.(H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.(I) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.


Figure 4 ( A ) Representative cross - sections of draining LNs ( dLNs ) from mice 7d post immunization with HEL - OVA / CFA following adoptive transfer of BHEL and transduced CD4OT - II cells . dLN were cryosectioned and stained with fluorophore - conjugated antibodies against GL - 7 to reveal GCs ( green ) , CD21 / 35 to stain for follicular dendritic cells ( FDC , red ) and Hoechst to stain for cell nuclei ( blue ) . Scale bar , 250µm . ( B , C ) Quantification of the number of GCs relative to B cell follicles ( B ) and size of GC ( C ) on confocal micrographs as shown in A . Shown are mean values with SD from dLNs from three mice in two independent experiment . Each dot represents one LN ( 3 LNs / mouse ) in B and one GC in C .

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Figure 4(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.


Figure 5 ( A ) Still images of the Movies EV3 ( Control , upper panel ) and EV4 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) , respectively . High endothelial venules ( HEVs ) ( white ) were labeled by intravenous injection of Alexa660 - labeled Meca - 79 antibody and collagen ( blue ) is visualized by second harmonic generation ( SHG ) . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( B ) Quantification of the duration of interactions as indicated by the arrows in A . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Data is derived from 5 to 6 movies , acquired from 4 mice in 2 independent experiments . Percentages on top indicate frequency of interactions that lasted 20min or longer . ( C ) Still images of the Movies EV5 ( Control , upper panel ) and EV6 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL cells ( green ) and mTomato CD4OT - II cells ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) . Follicular dendritic cells ( FDCs ) ( white ) were labeled by subcutaneous injection of Alexa647 - labeled anti - CD21 / CD35 antibody and collagen ( blue ) is visualized by SHG . Yellow and orange arrows indicate stable and transient ISs , respectively . Numbering of ISs corresponds to that in the respective Supplemental movies . Scale bar 20µm . ( D ) Quantification of the duration of interactions as indicated by the arrows in C . Touching T and B cells were manually quantification using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Shown is one representative dataset out of two , derived from 5 to 8 movies , acquired from 4 mice . Percentages on top indicate frequency of interactions that lasted 20min or longer .

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

Figure 5(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer. (C) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer.


Figure 6 ( B ) Virus replication as assessed by quantification of RT activity in the cell culture supernatant . Shown are mean values with SD from 6 donors . ( C ) Production of IgG antibodies against TT from infected blocks at indicated time point . Each dot represents one donor , bar indicates mean value . Tonsillar blocks were infected with indicated virus for 24h . After washing off the virus , blocks were stimulated with TT antigen for 24h . Samples were collected at different time intervals to estimate viral replication and antibody production .

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Figure 6(B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors.(C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.


Figure 1Comparison of structural preservation in cryosections picked up with sucrose ( a ) and MU ( b ) . In isolated hepatocytes , autophagy was induced for 30 min in suspension buffer before fixation . ( a ) Cryosection picked up with 2 . 3 M sucrose and labeled for SOD . Note the presence of large , " empty " gaps between sequestered contents of the AV and the surrounding cytoplasm . ( b ) A similar section as a , but picked up with an MU mixture . The AV are no longer distorted and the overall membranous structures are sharply delineated . The SOD - labeling density of AVi is as that of the cytosol , whereas AVd show much more intense labeling . ( Inset ) An AVi / d that is distinct from AVi by its single limiting membrane . The highly pleiomorphic AVd are also bound by a distinctive single membrane , but in contrast to AVi / d , their contents are characterized by debris of digested material . Bars , 250 nm .

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Figure 1Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, "empty" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.


Figure 2Labeling patterns of cathepsin D and CAIII in MU picked - up cryosections . ( a ) Cells treated as in Fig . 1 b , but the section was immunolabeled for cathepsin D , which is not detectable in the AVi , moderately present in the AVi / d , and abundant in AVd . Cells in b and c had endocytosed 5 - nm gold tracer for 20 and 40 min , respectively , before fixation and CAIII labeling . ( b ) Complex fusion profile of a CAIII - positive AVi , an AVd which is without CAIII labeling , and a VE that contains an endocytosed gold probe ( arrow ) . Close to the AVd is another tracer - bearing VE . ( c ) Fusion profile of a tracer - bearing MVE and an AVi / d that exhibits a single limiting membrane and a CAIII - labeling concentration similar to that of the cytoplasm . Bars , 200 nm .

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Figure 2Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.


Figure 3Appearance of tracer in AV . Cells were allowed to endocytose tracer particles continuously for the designated periods of time . At each time , random sections were searched for AV + , and AV + / 500 cell profiles were plotted . The number of cell profiles examined ( denominator ) and the respective number of AV + scored ( numerator ) are listed above the graph .

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Figure 3Appearance of tracer in AV. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+, and AV+/500 cell profiles were plotted. The number of cell profiles examined (denominator) and the respective number of AV+ scored (numerator) are listed above the graph.


Figure 4Tracer distribution in AV + types . Cells were allowed to endocytose tracer particles continuously for the designated periods of time . At each time , random sections were searched for AV + . The results are categorized according to AV types and plotted for the relative distribution of tracers . The total tracer counts are given on top of each bar . The number in each block represents the actual number of AV profiles , from which tracer counts are scored .

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Figure 4Tracer distribution in AV+ types. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+. The results are categorized according to AV types and plotted for the relative distribution of tracers. The total tracer counts are given on top of each bar. The number in each block represents the actual number of AV profiles, from which tracer counts are scored.


Figure 5Fusion profiles . Cells exposed to 5 - nm gold tracers for 20 ( a and c ) and 25 min ( b ) before fixation . Sections were picked up by MU and air - dried ( c ) or underwent immunolabeling with anti - SOD antibodies ( a and b ) . ( a ) Fusion between a small VE ( arrow ) and an AVi . A cluster of two gold particles ( arrowhead ) is also seen at another location . ( b ) Fusion between an electron lucent MVE ( arrow ) and an AVd . ( c ) Fusion between an MVE ( arrow ) and an AVd . Bars , 200 nm .

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Figure 5Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.


Figure 6Endosome types involved in FP + . The FP + were classified according to MVE and VE involved . The actual numbers of FP + analyzed are given above the bars .

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Figure 6Endosome types involved in FP+. The FP+ were classified according to MVE and VE involved. The actual numbers of FP+ analyzed are given above the bars.


Figure 7ASGP - R labeling in FP + and endosomes . Cells exposed for 40 min to 5 - nm gold tracer . Section immunolabeled for ASGP - R with 10 - nm gold . ( a ) Low ASGP - R labeling in tracer - carrying endocytic vesicles ( arrowheads ) , adjacent to ( top ) or fusing with ( bottom ) an AVi / d . ( b ) ASGP - R labeling at the plasma membrane and in endosomal elements near the cell surface ( ex , extra cellular space ) . Vesicles of the MVE ( asterisk ) and VE ( arrowhead ) type , containing endocytosed gold , are present in the same area . ( c ) As b . A VE - like vesicle with endocytosed gold and low ASGP - R labeling ( arrowhead ) is shown connected to an ASGP - R rich element ( arrow ) , possibly a recycling endosome . Bars , 200 nm .

Figure 7ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.


Figure 8AV types involved in FP + . The same FP + data as used in Fig . 6 were classified according to the AV types .

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Figure 8AV types involved in FP+. The same FP+ data as used in Fig. 6 were classified according to the AV types.


figf1 ( b ) PC12 cells transfected with either 60Q or 60Q - HSC70bm . The presence of two HSC70bm motifs in 60Q - HSC70bm reduced the number of inclusions as compared to 60Q ( scale bar , 20 μm ) . ( c ) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q - HSC70bm . 10 mM 3 - methyladenine ( 3 - MA ) , 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy , the proteases , cathepsins D and E , and cathepsin A , respectively . Bars in c represent relative mean values ± s . e . m . from four independent experiments , with levels of aggregation observed for 60Q normalized to a value of 1 . Quantifications were performed by ArrayScan .

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figf1(b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.


figf2 ( b ) Confocal images of the 150Q Neuro2a cells transfected with R , RH , RS , RHS , RQ and RHQ . Molecules containing QBP1 co - localized with the tNHTT - 150Q - EGFP inclusions ( bar , 5 μm ) . Cells with inclusions were chosen to illustrate the co - localization of QBP1 - containing molecules with tNHTT - 150Q - EGFP . ( c ) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT - 150Q - EGFP on the gel top . Note the shift of tNHTT - 150Q - EGFP from an insoluble to a soluble form when RQ was co - expressed . The levels of tNHTT - 16Q - EGFP were not affected . Full - length western blots are presented in Supplementary Figure 13 . ( d ) RHQ reduced the levels of aggregated and soluble forms of tNHTT - 150Q - EGFP by 81 . 8 % and 33 . 6 % , respectively . WB , western blot . ( e ) Quantification of tNHTT - 16Q - EGFP protein levels . ( f ) ArrayScan analysis revealed 28 % and 56 % reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ , respectively , after 24 h of differentiation and induction . ( g ) In 150Q - nls Neuro2a cells , expression of RQ and RHQ reduced the number of tNHTT - 150Q - nls - EGFP inclusion by 23 % ( RQ ) and 53 % ( RHQ ) after 48 h of differentiation and induction compared to RS and RHS , respectively . Bars in d and e represent mean values ± s . e . m . from four independent experiments . Quantifications were performed by ArrayScan .

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figf2(b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP.(c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13.(d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels.(f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.


figf3 ( a ) Western blot analysis of the chase experiment in 60Q Neuro2a cells . Full - length western blots are presented in Supplementary Figure 13 . ( b ) RHQ enhanced the tNHTT - 60Q - EGFP degradation during the chase period . 5 μM MG132 , 10 mM 3 - methyladenine and 15 mM ammonium chloride ( NH4Cl ) were used to inhibit proteasome , macroautophagy and lysosomal degradation , respectively . NH4Cl had no effect on proteasome activity and did not affect cell viability ( Supplementary Fig . 4a , b ) . Bars in b represent relative mean values ± s . e . m . from three independent experiments . Protein levels at time 0 represent the control condition ( arbitrarily set as 1 ) .

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

figf3(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).