( a ) Mean number of autophagosomes per motor neuron field in electron micrographs from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 0 . 63 , one - way ANOVA with post hoc Tukey test . P = 0 . 534 . 14 months : n = 3 independent experiments , F = 4 . 32 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( b ) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 4 . 18 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . 14 months : n = 3 independent experiments , F = 4 . 56 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( c ) Mean autophagy index , dividing number of autolysosome by number of autophagosomes for each motor neuron field , ± s . e . m . At 6 months , YAC AR100 motor neurons displayed a markedly increased autophagy index ; n = 3 independent experiments , F = 129 . 81 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . However , once YAC AR100 mice develop disease pathology at 14 months , the autophagy index for YAC AR100 motor neurons is significantly decreased ; n = 3 independent experiments , F = 82 . 82 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(a) Mean number of autophagosomes per motor neuron field in electron micrographs from non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 0.63, one-way ANOVA with post hoc Tukey test. P = 0.534. 14 months: n = 3 independent experiments, F = 4.32, one-way ANOVA with post hoc Tukey test. *P 0.05. (b) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt, YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 4.18, one-way ANOVA with post hoc Tukey test. *P 0.05. 14 months: n = 3 independent experiments, F = 4.56, one-way ANOVA with post hoc Tukey test. *P 0.05. (c) Mean autophagy index, dividing number of autolysosome by number of autophagosomes for each motor neuron field, ± s.e.m. At 6 months, YAC AR100 motor neurons displayed a markedly increased autophagy index; n = 3 independent experiments, F = 129.81, one-way ANOVA with post hoc Tukey test. **P 0.01. However, once YAC AR100 mice develop disease pathology at 14 months, the autophagy index for YAC AR100 motor neurons is significantly decreased; n = 3 independent experiments, F = 82.82, one-way ANOVA with post hoc Tukey test. **P 0.01. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) HEK293 cells were transfected with TFEB and AR25Q or AR125Q , as indicated . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

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

(a) HEK293 cells were transfected with TFEB and AR25Q or AR125Q, as indicated. Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( b ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation . Untreated : n = 3 independent experiments , F = 3 . 48 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Sucrose treatment : n = 3 independent experiments , * * P 0 . 01 , t - test , WT t ( 10 ) = 8 . 133 ; * * * P 0 . 001 , t - test , AR 24Q t ( 10 ) = 6 . 11 ; P not significant ( n . s . ) , AR 65Q t ( 10 ) = 0 . 644 . Sucrose - treated MN - 1 AR24Q cells displayed significantly higher 4X - CLEAR luciferase activity : n = 3 independent experiments , F = 2 . 49 , one - way ANOVA with post hoc Tukey test . * * * P 0 . 001 . All ratios were normalized to untreated MN - 1 WT cells , whose value was set to 1 .

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

(b) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation. Untreated: n = 3 independent experiments, F = 3.48, one-way ANOVA with post hoc Tukey test. **P 0.01. Sucrose treatment: n = 3 independent experiments, **P 0.01, t-test, WT t(10) = 8.133; ***P 0.001, t-test, AR 24Q t(10) = 6.11; P not significant (n.s.), AR 65Q t(10) = 0.644. Sucrose-treated MN-1 AR24Q cells displayed significantly higher 4X-CLEAR luciferase activity: n = 3 independent experiments, F = 2.49, one-way ANOVA with post hoc Tukey test. ***P 0.001. All ratios were normalized to untreated MN-1 WT cells, whose value was set to 1.


( c ) We exposed MN - 1 cells to ammonium chloride , an inhibitor of lysosomal activity , to determine the capacity for TFEB induction . RT - PCR analysis revealed lack of TFEB target gene induction in MN - 1 AR65Q cells for four gene targets : Lamp1 ( lysosomal - associated membrane protein 1 ) , F = 37 . 51 ; Atp6v1h ( vesicular ATPase V1 subunit H ) , F = 57 . 26 ; Mcoln1 ( mucolipin 1 ) , F = 251 . 0 ; and Gla ( galactosidase - α ) , F = 75 . 51 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , ANOVA with post hoc Tukey test . For Lamp1 and Mcoln1 , MN - 1 AR24Q cells yielded significantly higher expression . Differences in the expression of TFEB target genes at baseline were negligible .

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

(c) We exposed MN-1 cells to ammonium chloride, an inhibitor of lysosomal activity, to determine the capacity for TFEB induction. RT-PCR analysis revealed lack of TFEB target gene induction in MN-1 AR65Q cells for four gene targets: Lamp1 (lysosomal-associated membrane protein 1), F = 37.51; Atp6v1h (vesicular ATPase V1 subunit H), F = 57.26; Mcoln1 (mucolipin 1), F = 251.0; and Gla (galactosidase-α), F = 75.51; n = 3 independent experiments, *P 0.05, **P 0.01, ANOVA with post hoc Tukey test. For Lamp1 and Mcoln1, MN-1 AR24Q cells yielded significantly higher expression. Differences in the expression of TFEB target genes at baseline were negligible.


( d ) We measured expression levels of TFEB target genes in E13 motor neurons derived from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 mice by RT - PCR analysis : Ctsf ( cathepsin F ) , F = 4 . 63 ; Mcoln1 , F = 6 . 64 ; Atp6v1h , F = 5 . 6 ; and Gla , F = 5 . 65 ; n = 3 independent experiments , * P 0 . 05 , ANOVA with post hoc Tukey test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(d) We measured expression levels of TFEB target genes in E13 motor neurons derived from non-transgenic (Nt), YAC AR20 and YAC AR100 mice by RT-PCR analysis: Ctsf (cathepsin F), F = 4.63; Mcoln1, F = 6.64; Atp6v1h, F = 5.6; and Gla, F = 5.65; n = 3 independent experiments, *P 0.05, ANOVA with post hoc Tukey test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and with BFP - tagged TFEB or BFP empty vector , and then measured luciferase activity . Marked induction of luciferase activity occurred with BFP - TFEB ; n = 3 independent experiments , * * * P 0 . 001 , t - test , WT t ( 4 ) = 48 . 79 ; * * * P 0 . 001 , t - test , AR 24Q t ( 4 ) = 22 . 91 ; * * * P 0 . 001 , t - test , AR 65Q t ( 4 ) = 14 . 02 . Results for each line were normalized to the BFP - empty luciferase activity .

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

(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity.


( b ) We transfected MN - 1 AR65Q cells with the 4X - CLEAR luciferase reporter and with BFP - TFEB or BFP empty vector and subjected the MN - 1 AR65Q cells to conditions that promote TFEB activation , as shown . MN - 1 WT cells were also transfected with the 4X - CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes . Results are shown normalized to untreated MN - 1 WT cells expressing BFP - empty . Untreated , F = 182 . 7 ; starvation , F = 63 . 64 ; NH4Cl , F = 291 . 9 ; Rapamycin , F = 3128 ; ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 .

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

(b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001.


( c ) We transfected MN - 1 AR65Q cells with the mCherry - EGFP - LC3 construct and with either BFP - empty or BFP - TFEB . Autophagic vesicles forming in BFP - expressing cells were then detected as red , green or yellow puncta , as shown . Scale bar , 20 μm . ( d ) Left , quantification of autophagic vesicle type . Note marked reduction in autophagosomes in MN - 1 AR65Q cells expressing BFP - TFEB ; n = 3 independent experiments , F = 16 . 596 , ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 . Right , calculation of the autophagy index ; n = 3 independent experiments , F = 24 . 43 , ANOVA with post hoc Tukey test . * * P 0 . 01 . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( b ) HEK293 cells were transfected with TFEB and influenza hemagglutinin ( HA ) - tagged full - length AR25 ( FL ) or a C - terminal AR mutant ( m1 , m2 , m3 , m4 ) . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( c ) HEK293 cells were transfected with TFEB and HA - tagged N - terminal AR mutant containing a 19Q or 112Q tract ( NT19Q , NT112Q ) . Immunoprecipitation was performed as in b . with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

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

(c) HEK293 cells were transfected with TFEB and HA-tagged N-terminal AR mutant containing a 19Q or 112Q tract (NT19Q, NT112Q). Immunoprecipitation was performed as in b. with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( d ) MN - 1 WT , MN - 1 AR24Q and MN - 1 AR65Q cells were cultured in standard medium ( Ctl ) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB . Cells were scored for nuclear or cytosolic localization of TFEB . Scale bars , 20 μm . ( e ) Quantification of d . Untreated , F = 0 . 56 ; R1881 , F = 5 . 51 ; ANOVA with post hoc Tukey test . * * P 0 . 01 .

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

(d) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01.


( f ) We transfected MN - 1 WT cells with the 4X - CLEAR luciferase reporter and either BFP - empty or AR25Q - BFP , under standard culture conditions ( control ) , ammonium chloride treatment or rapamycin treatment . Control , t ( 3 ) = 15 . 73 ; NH4Cl , t ( 4 ) = 4 . 108 ; rapamycin . t ( 4 ) = 4 . 58 ; t - test . * * P 0 . 01 .

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

(f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01.


( g ) We transfected MN - 1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X - CLEAR luciferase reporter activity under baseline conditions ( control ) or after rapamycin . Control , t ( 4 ) = 0 . 51 ; rapamycin , t ( 4 ) = 7 . 15 ; t - test . * * P 0 . 01 .

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

(g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01.


( h ) RT - PCR analysis of MN - 1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector : Gla , t ( 4 ) = 4 . 77 ; Atp6v1h , t ( 4 ) = 3 . 75 ; Mcoln1 , t ( 4 ) = 13 . 74 ; Ctsd ( cathepsin D ) , t ( 4 ) = 7 . 91 ; and Lamp1 , t ( 4 ) = 6 . 46 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , t - test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(h) RT-PCR analysis of MN-1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector: Gla, t(4) = 4.77; Atp6v1h, t(4) = 3.75; Mcoln1, t(4) = 13.74; Ctsd (cathepsin D), t(4) = 7.91; and Lamp1, t(4) = 6.46; n = 3 independent experiments, *P 0.05, **P 0.01, t-test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We performed filter - trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients . Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines , as based on densitometry ; n = 3 independent experiments , P 0 . 05 , t - test , t ( 4 ) = 4 . 026 . AU , arbitrary units .

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

(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units.


( b ) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC - 1 dye . Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 4 ) = 3 . 983 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983.


( c ) We transfected NPC lines derived from SBMA patients and controls ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector . Autophagic vesicles were detected as colored puncta , as shown . Scale bar , 10 μm . ( d ) Quantification of autophagic vesicle type from c . SBMA NPCs exhibit a significant increase in autophagosomes ; n = 18 different samples : 3 clones per line , 3 lines per group , * P 0 . 05 , t - test ; t ( 18 ) = 2 . 3799 ( autophagosomes ) .

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

(c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes).


( e ) We isolated RNAs from SBMA and control NPC lines ( three different NPC lines per human subject ) and then measured the expression levels of TFEB target genes including CYCS ( cytochrome c ) , t ( 4 ) = 2 . 844 ; MCOLN1 , t ( 4 ) = 3 . 246 ; GLA , t ( 4 ) = 3 . 174 ; ACADM ( acyl - CoA dehydrogenase ) , t ( 4 ) = 4 . 405 ; COX6A1 ( cytochrome oxidase 6A1 ) , t ( 4 ) = 5 . 584 ; and the TFEB gene itself , t ( 4 ) = 0 . 593 . Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes . * P 0 . 05 , * * P 0 . 01 . Data in d , e are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone ( DHT ) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation ( IP ) . Immunoblotting ( IB ) for AR confirmed expression of AR in control ( Ctrl ) and SBMA NPCs , and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs .

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

(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs.


( b ) We transfected SBMA NPC lines ( three different NPC lines per patient ) with either the BFP - empty vector or BFP - TFEB expression construct , treated the NPCs with JC - 1 dye and counted the percentage of cells with depolarized mitochondria . Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 6 ) = 3 . 217 .

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(b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217.


( c ) Representative images from transfection of SBMANPCs with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct . BFP - empty vector - expressing SBMANPCs display a preponderance of yellow puncta ( autophagosomes ) , while BFP - TFEB - expressing SBMANPCs exhibit a shift toward almost entirely red puncta ( autolysosomes ) , consistent with rescue of the autophagic flux defect . Scale bars , 20 μm . ( d ) We transfected SBMANPC lines ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct , counted the number of autophagosomes and autolysosomes in BFP - expressing NPCs and determined the number of autophagic vesicles per cell . TFEB overexpression markedly reduced autophagosome formation in SBMANPCs . * * * P 0 . 001 ; t - test , t ( 30 ) = 4 . 373 . Data in d are presented as mean ± s . e . m . Individual P values are available in the Supplementary Methods Checklist .

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

(c) Representative images from transfection of SBMANPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector-expressing SBMANPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB-expressing SBMANPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMANPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMANPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.


C . Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are based on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral counts ( normalized plus pseudocounts ) ± SEM , n = 3 independent biological repeats . All proteins shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 and a mean fold change > 2 by SAINTexpress analysis

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

C. Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass-spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis


C . Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are based on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral counts ( normalized plus pseudocounts ) ± SEM , n = 3 independent biological repeats . All proteins shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 and a mean fold change > 2 by SAINTexpress

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


A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation analysis of Kif5a and HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 cells ) . IP was performed with HA antibody and probed in Western blot with anti - Kif5a and anti - HA antibodies . Ncl FL - HA - Dendra2 - full length nucleolin ; Ncl ΔGAR - HA - Dendra2 - nucleolin with a GAR domain deletion ; Ncl GAR ( N ) - HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ; DEN - Dendra2 . Note that HA input blots for HA - Dendra2 ( Dendra ) and the other three constructs are from the same membrane , but shown discontinuously owing to the different migration of these proteins in PAGE .

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

A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.


A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation analysis of Kif5a and HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 cells ) . IP was performed with HA antibody and probed in Western blot with anti - Kif5a and anti - HA antibodies . Ncl FL -

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

A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA


D . Uptake of TAMRA - labeled R4 and N4 peptides into cultured DRG neurons . Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed and analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm . E . Quantification of ( D ) . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , paired Student ' s t - test a . u . - arbitrary units .

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

D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm. E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units.


D . Uptake of TAMRA - labeled R4 and N4 peptides into cultured DRG neurons . Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed and analyzed by confocal microscopy . PC - phase contrast . 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

D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm.


G . HEK - 293 cells were transfected with plasmids expressing HA - Dendra2 - full length nucleolin ( Ncl FL ) , HA - Dendra2 - nucleolin with a GAR domain deletion ( Ncl ΔGAR ) or HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ( Ncl GAR ( N ) ) and processed as in ( F ) .

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

G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).


G . HEK - 293 cells were transfected with plasmids expressing HA - Dendra2 - full length nucleolin (

G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl


A . GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons , transduced with the peripheral neuron specific AAV - PHP . S vector expressing full length HA - Dendra2 - nucleolin ( Ncl FL ) or respective ΔGAR mutant ( Ncl ΔGAR ) upon seeding and analyzed by epifluorescence microscopy 9 - 10 days later . Shown are representative Dendra2 fluorescence images collected from the non - activated ( green ) emission line . Hoechst 33342 ( 10 µM ) was used to outline nuclei . Superimposed phase contrast and Dendra2 fluorescence images are shown on the right . Scale bar - 25 µm . B . Quantification of the nucleolar / nuclear intensity ratios in DRG cells shown in ( A ) . n = 38 - 54 cells per sample in 3 independent biological repeats ; means ± SEM ; * * * * p < 0 . 0001 , Mann - Whitney 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

A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm. B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test


C . Time - lapse live imaging of nuclei in DRG neurons transduced with AAV - PHP . S HA - Dendra2 - nucleolin ( full length and ΔGAR mutant ) , after a hypotonic challenge with double - distilled water ( ddH20 ) . Shown are epifluorescence images ( Dendra2 green emission line ) taken at indicated times after medium replacement with ddH20 . Scale bar - 10 µm . D . Quantification of nucleolar Dendra2 signal in ( C ) with intensity values immediately before medium replacement set as 100 % . n = 17 - 24 cells per sample in 3 biological repeats ; means ± SEM ; Two - way ANOVA with Sidák ' s post - test detected a significant time - dependent reduction of nucleolar signal ( p < 0 . 0001 ) and no difference between full length and ΔGAR mutant nucleolin ( p > 0 . 05 )

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

C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm. D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05)


B . Western blot analysis of nucleolin in DRG neurons from wildtype ( WT ) and GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . The higher nucleolin band in GAR + / - corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in protein size ) . C . Quantification of ( B ) , total nucleolin levels ( sum of both bands where applicable ) were normalized to Tuj1 and expressed as GAR + / - / WT ratios . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , 1 sample t - test .

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

B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size). C. Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR+/- / WT ratios. n = 3 independent biological repeats; means ± SEM; * p < 0.05, 1 sample t-test.


B . Western blot analysis of nucleolin in DRG neurons from wildtype ( WT ) and GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . The higher nucleolin band in GAR + / - corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in protein size ) .

B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).


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 .

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

A, 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 .

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.


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 .

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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.


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

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).


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 .

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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.


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 .

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

A. 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.


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

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).


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 .

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

C. 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.


Total 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

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

Total 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 mutations


Large ( > 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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Large (>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


Allele 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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Allele 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


Unsupervised 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 %

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Unsupervised 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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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


TSS enrichment score of primary samples ( PRI ) and

TSS enrichment score of primary samples (PRI) and PDXs


Reads number per peak in primary samples ( x - axis ) plotted against the reads number in corresponding PDX model ; PCC = Pearson ' s correlation coefficient )

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Reads 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 )

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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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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


( 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 ) .

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(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 .

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(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 .

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


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

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


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

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(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 .

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


( A ) SKO 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 ) .

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


( C ) 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 .

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(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 ) .

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(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 ) .

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


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

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(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 ) .

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


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

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


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

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


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

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(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 ) .

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


( G ) 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 .

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


( A ) WT 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 .

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(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 .

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(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 ) .

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(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 ) .

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


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

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


( B ) WT 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 .

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


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

(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 ) .

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(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 ) .

(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 .

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


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

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(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 ) .

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


( D ) Control 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 ) .

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


( F ) Control 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 ) .

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(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 .

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


( A ) Western blot analysis for pRb in DM - 2 cultures .

(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 .

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(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 ) .

(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 ) .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 ) .

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(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).