figf1 ( a ) MN - 1 cells were transfected with mCherry - EGFP - LC3 vector , fixed and imaged after 24 h . Numbers of yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) per cell were counted . Autophagosomes : n = 3 independent experiments , F = 3 . 56 ; autolysosomes : n = 3 independent experiments , F = 20 . 13 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * * P 0 . 001 . n = 34 cells per genotype . ( b ) We transfected MEFs stably expressing mCherry - EGFP - LC3 with BFP - tagged AR25Q or AR125Q . Cells were imaged after 24 h , and yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) in AR - expressing ( blue ) cells were counted . Autophagosomes : n = 3 independent experiments , F = 20 . 65 ; autolysosomes : n = 3 independent experiments , F = 4 . 49 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . n = 33 cells per genotype . ( c ) MN - 1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux . A representative LC3 western blot is shown . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( d ) The ratio of LC3 - II : actin for c determined by densitometry analysis using ImageJ . n = 3 independent experiments , F = 0 . 4501 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( e ) MN - 1 cells were treated with ammonium chloride to block lysosomal activity , and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6 - h recovery period . We performed p62 western blot analysis on MN - 1 cells at baseline ( - ) , after ammonium chloride treatment ( + ) and after the recovery period ( Rec ) . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( f ) On the basis of the p62 values obtained in e , we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline / recovery values to baseline / recovery for MN - 1 WT cells . n = 3 independent experiments , F = 5 . 45 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 . Data are presented as mean ± s . e . m . Scale bar , 20 μm . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

figf1(a) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h. Numbers of yellow puncta (autophagosomes) and red puncta (autolysosomes) per cell were counted. Autophagosomes: n = 3 independent experiments, F = 3.56; autolysosomes: n = 3 independent experiments, F = 20.13. One-way ANOVA with post hoc Tukey test. *P 0.05, ***P 0.001. n = 34 cells per genotype.(b) We transfected MEFs stably expressing mCherry-EGFP-LC3 with BFP-tagged AR25Q or AR125Q. Cells were imaged after 24 h, and yellow puncta (autophagosomes) and red puncta (autolysosomes) in AR-expressing (blue) cells were counted. Autophagosomes: n = 3 independent experiments, F = 20.65; autolysosomes: n = 3 independent experiments, F = 4.49. One-way ANOVA with post hoc Tukey test. *P 0.05, **P 0.01, ***P 0.001. n = 33 cells per genotype.(c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.(e) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf2 ( a , b ) Electron micrographs of motor neuron perinuclear regions from age - matched non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice before disease onset ( a , 6 months of age ) and after prominent neuromuscular and molecular pathology is apparent ( b , 14 months of age ) . ( a ) At 6 months of age , occasional autophagosomes ( yellow arrowheads ) are noted in Nt , YAC AR20 and YAC AR100 at roughly equivalent frequency . Autolysosomes ( red arrowheads ) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons . ( b ) At 14 months of age , when YAC AR100 mice display signs of motor neuronopathy and molecular pathology , we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes ( yellow arrowheads ) . Despite this increase in autophagosomes , autolysosomes were fewer in number at this age in YAC AR100 motor neurons , such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons . This was never the case for Nt motor neurons or YAC AR20 motor neurons . Main panels are at original magnification 2 , 200 × and insets are at original magnification 3 , 700 × .

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

figf2(a,b) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age). (a) At 6 months of age, occasional autophagosomes (yellow arrowheads) are noted in Nt, YAC AR20 and YAC AR100 at roughly equivalent frequency. Autolysosomes (red arrowheads) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons. (b) At 14 months of age, when YAC AR100 mice display signs of motor neuronopathy and molecular pathology, we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes (yellow arrowheads). Despite this increase in autophagosomes, autolysosomes were fewer in number at this age in YAC AR100 motor neurons, such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons. This was never the case for Nt motor neurons or YAC AR20 motor neurons. Main panels are at original magnification 2,200× and insets are at original magnification 3,700×.


figf3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) 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.


figf4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(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.(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.


figf5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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.(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.


figf6 ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(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).(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.(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.


figf7 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(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.(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.


figf8 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(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.(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.


Figure 1Detection of NRPB2WT - FLAG , NRPB2P979S - FLAG and NRPB2Y732F - FLAG protein by western blotting in NRPB2WT - FLAG Col - 0 , NRPB2P979S - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 plants . Untagged NRPB2 ( Col - 0 ) was used as a negative control . Histone H3 was used as an internal control and total protein level detected by stain - free blot was used as a loading control . Quantification was done by normalizing to the loading control and anti - H3 blot based on 3 independent replicates . Transmission rate of nrpb2 - 2 allele in nrpb2 - 2 + / - line ( n = 197 ) and nrpb2 - + / - lines combined with homozygous NRPB2P979S - FLAG + / + ( n = 280 ) , NRPB2Y732F - FLAG + / + ( n = 240 ) and NRPB2WT - FLAG + / + ( n = 210 ) , respectively . Fisher ´ s exact test was used as a statistic test , three asterisks denote p < 0 . 001 between samples and n . s . stands for not significant . Image of homozygous mutant nrpb2 - 2 fully complemented by NRPB2WT - FLAG ( top , NRPB2WT + / + nrpb2 - 2 - / - ) and partially complemented by NRPB2Y732F - FLAG ( bottom , NRPB2WT + / + nrpb2 - 2 - / - ) . Plants were grown for 4 weeks in soil . Scale bars represent 1 cm .

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

Figure 1Detection of NRPB2WT-FLAG, NRPB2P979S-FLAG and NRPB2Y732F-FLAG protein by western blotting in NRPB2WT-FLAG Col-0, NRPB2P979S-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 plants. Untagged NRPB2 (Col-0) was used as a negative control. Histone H3 was used as an internal control and total protein level detected by stain-free blot was used as a loading control. Quantification was done by normalizing to the loading control and anti-H3 blot based on 3 independent replicates.Transmission rate of nrpb2-2 allele in nrpb2-2 +/- line (n=197) and nrpb2- +/- lines combined with homozygous NRPB2P979S-FLAG +/+ (n=280), NRPB2Y732F-FLAG +/+ (n=240) and NRPB2WT-FLAG +/+ (n=210), respectively. Fisher´s exact test was used as a statistic test, three asterisks denote p<0.001 between samples and n.s. stands for not significant.Image of homozygous mutant nrpb2-2 fully complemented by NRPB2WT-FLAG (top, NRPB2WT +/+ nrpb2-2 -/-) and partially complemented by NRPB2Y732F-FLAG (bottom, NRPB2WT +/+ nrpb2-2 -/-). Plants were grown for 4 weeks in soil. Scale bars represent 1 cm.


Figure 2Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo . In brief , Arabidopsis seedlings of NRPB2WT - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days . Flagellin peptides ( flagellin 22 ) were added into media and treated samples were collected in a 0 minute ( no treatment ) , 2 minutes and 4 minutes time course . The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes . See Methods for technical details . Nascent RNA profile of AT5G41750 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represent 0 . 5 kb . Nascent RNA profile of AT4G19520 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represents 0 . 5 kb .

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

Figure 2Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details.Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb.Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.


Figure 3plaNET - seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal at the region of promoter - proximal stalling . Metagene profile of plaNET - seq mean signal of RNAPII in a 1 Kb window centered at the + 1 nucleosome in Arabidopsis genes ( n = 25474 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from - 25 bins to + 25 bins around + 1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two - sided Mann - Whitney U - test , p = 5 . 20e - 10 . RNAPII stalling index calculated for all the genes with plaNET - Seq FPKM ≥ 10 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) ( n = 6596 ) . Medians of the stalling index are 1 . 891 and 1 . 222 for NRPB2WT and NRPB2Y732F , respectively . * * * denotes p - value < 0 . 001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Metagene profile of plaNET - seq mean signal over whole genes ( length from 0 . 5 Kb to 5 Kb , scaled to 500 bins , n = 27042 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Metagene profile of plaNET - seq mean signal of RNAPII in exons ( length from 50 bp to 300 bp , scaled to 100 bins , n = 73925 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in exons , p < 1e - 16 . Metagene profile of plaNET - seq mean signal of RNAPII in introns ( 50 bp to 300 bp , scaled to 100 bins , n = 102260 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in introns , p < 1e - 16 .

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

Figure 3plaNET-seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal at the region of promoter-proximal stalling.Metagene profile of plaNET-seq mean signal of RNAPII in a 1 Kb window centered at the +1 nucleosome in Arabidopsis genes (n=25474) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from -25 bins to +25 bins around +1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two-sided Mann-Whitney U-test, p=5.20e-10.RNAPII stalling index calculated for all the genes with plaNET-Seq FPKM ≥ 10 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red) (n=6596). Medians of the stalling index are 1.891 and 1.222 for NRPB2WT and NRPB2Y732F, respectively. *** denotes p-value <0.001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Metagene profile of plaNET-seq mean signal over whole genes (length from 0.5 Kb to 5 Kb, scaled to 500 bins, n=27042) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Metagene profile of plaNET-seq mean signal of RNAPII in exons (length from 50 bp to 300 bp, scaled to 100 bins, n=73925) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons. A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in exons, p<1e-16. Metagene profile of plaNET-seq mean signal of RNAPII in introns (50 bp to 300 bp, scaled to 100 bins, n=102260) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in introns, p<1e-16.


Figure 4Bar charts showing the fractions of 3 ' and 5 ' splicing intermediate reads from plaNET - seq in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . The fraction of RNA - seq intronic reads calculated for all genes ( n = 24912 ) in NRPB2WT + / + nrpb2 - 2 - / - and NRPB2Y732F + / + nrpb2 - 2 - / - . Two - sided Mann - Whitney U test : * * * * denotes p - value < 2 . 2e - 16 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Differentially expressed ( DE ) exons and introns in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - based on RNA - seq results . Numbers of DE exons and introns were shown in plot . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) exons and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * denotes p - value < 0 . 01 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) introns and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * * * denotes p - value < 0 . 0001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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

Figure 4Bar charts showing the fractions of 3' and 5' splicing intermediate reads from plaNET-seq in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb.The fraction of RNA-seq intronic reads calculated for all genes (n=24912) in NRPB2WT +/+ nrpb2-2 -/- and NRPB2Y732F +/+ nrpb2-2 -/-. Two-sided Mann-Whitney U test: **** denotes p-value<2.2e-16. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Differentially expressed (DE) exons and introns in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/- based on RNA-seq results. Numbers of DE exons and introns were shown in plot. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) exons and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. ** denotes p-value <0.01 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) introns and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. **** denotes p-value <0.0001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Figure 5plaNET - seq signal of RNAPII at 3 ' end of AT2G21410 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal peaks at PAS stalling region . Metagene profile of plaNET - seq mean signal of RNAPII in a 1 kb window centered at PAS ( n = 24448 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from PAS to + 100 bins between NRPB2WT and NRPB2Y732F were assessed by Two - sided Mann - Whitney U - test , p = 1 . 53e - 06 . Histogram of transcriptional read - through length ( nt ) from PAS of protein - coding gene ( plaNET - seq FPKM > 5 , n = 9316 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Box plot shows the RNAPII transcriptional read - through length from PAS of protein - coding genes ( plaNET - seq FPKM > 5 n = 9316 ) called based on statistic model ( see Methods ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Median of read - through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt . Two - sided Mann - Whitney U - test : * * * denotes p = 9 . 9e - 62 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile . Metagene plot of RNAPII signal by plaNET - seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes ( n = 5753 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrow denotes the direction of transcriptional read - through . Pink dashed line rectangle indicates the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction . Metagene plot of RNAPII signal by plaNET - seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in " tail to tail " orientation ( n = 1384 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrows denote the directions of transcriptional read - through from both PASs . Pink dashed line rectangles indicate the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction .

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

Figure 5plaNET-seq signal of RNAPII at 3' end of AT2G21410 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal peaks at PAS stalling region.Metagene profile of plaNET-seq mean signal of RNAPII in a 1 kb window centered at PAS (n=24448) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from PAS to +100 bins between NRPB2WT and NRPB2Y732F were assessed by Two-sided Mann-Whitney U-test, p = 1.53e-06.Histogram of transcriptional read-through length (nt) from PAS of protein-coding gene (plaNET-seq FPKM>5, n=9316) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).Box plot shows the RNAPII transcriptional read-through length from PAS of protein-coding genes (plaNET-seq FPKM>5 n=9316) called based on statistic model (see Methods) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Median of read-through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt. Two-sided Mann-Whitney U-test: *** denotes p = 9.9e-62. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.Metagene plot of RNAPII signal by plaNET-seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes (n=5753) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrow denotes the direction of transcriptional read-through. Pink dashed line rectangle indicates the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction. Metagene plot of RNAPII signal by plaNET-seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in "tail to tail" orientation (n=1384) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrows denote the directions of transcriptional read-through from both PASs. Pink dashed line rectangles indicate the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction.


Figure 1A . Binding of ubiquitin chains to immobilized GST - fusion proteins . Indicated proteins were purified from E . coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains , which were either K48 - or K63 - linked . The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS ‑ PAGE and fluorescence scan . Total fluorescence was quantified per lane and normalized to the first lane ( rel . fluo . ) . Asterisk ( * ) indicates lane from the same gel that was cropped and moved .

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

Figure 1A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.


Figure 2B . At indicated time points , samples were taken from the reaction mix and analyzed by SDS ‑ PAGE and fluorescence scan . Product fluorescence over total fluorescence per lane was quantified ( rel . fluo . ) . D . Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full ‑ length Ubc1 or Ubc1 - ΔUBA ( aa1 ‑ 150 ) . The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin , while acceptor sites were blocked by introducing the K48R substitution . Reactions were performed in triplicate .

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Figure 2B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.


Figure 3b . L143E and / or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC / UBA domain interface . Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63 ‑ Ub2 with Ub ( K48R ) at the distal position or monoubiquitin as acceptor . Experiment and initial rate determinationc . Ubc1 variants were generated with deletions in the linker region ( aa152 ‑ 167 ) , which connects the UBA domain and the UBC domain . Single turnover ubiquitination reaction mixes were incubated for 5 min with K63 - Ub2 or for 20 min with monoubiquitin as acceptor . Final product formation was analyzed by SDS - PAGE and fluorescence scan . Product fluorescence over total fluorescence ( rel . fluo . ) was quantified . d . C - terminally blocked M1 - linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full - length Ubc1 or Ubc1 - ΔUBA . Reactions with acceptor K63 - Ub2 were carried out controls . Initial rate

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Figure 3b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determinationc. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination


Figure 4e . CSPs of I44 were plotted over UBA domain concentration . f . KD values for binding of the UBA domain were determined from results in ( E ) assuming two independent but different binding sites .

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Figure 4e. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.


Figure 5a . The activity of Ubc1 was compared with the K48 - linked chain building enzyme Ubc7 / Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin , K48 ‑ Ub2 or K63 - Ub2 . Initial rates were determinedb . The K63 - linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K ( h . sapiens ) were employed in single turnover ubiquitination experiments in presence of K63 - Ub2 with Ub ( K48R ) at the proximal or distal position or in presence of K48 ‑ Ub2 . Initial rate determination c . Experiment as in ( B ) but with the Ubc1 homologue ubc - 20 ( c . elegans ) .

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Figure 5a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determinedb. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).


Figure 6D . Alexa488 - labeled monoubiquitin and C - terminally capped K63 - Ub4 were used in a 4 : 1 molar ratio for in vitro ubiquitination reactions with full ‑ length Ubc1 or Ubc1 - ΔUBA . Where indicated , Alexa488 - labeled monoubiquitin was methylated prior to the reaction ( met - Ub ) to block de novo chain assembly . Reactions were incubated overnight , quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH ( lanes 4 - 6 ) . Samples were analyzed by SDS - PAGE and fluorescence scan .

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Figure 6D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.


Figure 7F . Ubc1 - EA was genomically integrated into various yeast strains . Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10 ‑ fold dilutions . Where noted cells were incubated at elevated temperature ( 37 ° C ) or in presence of hydroxyurea ( HU ) to induce DNA replication stress .

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Figure 7F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.


Figure 1CSF extract was treated with DMSO , CsA , buffer or His - CnA420 - 508 at the indicated concentrations . Meiotic exit was induced by calcium addition and samples were taken at the indicated time points . Samples were immunoblotted for cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was treated with DMSO or CsA . Both reactions were divided and supplemented with the indicated amounts of calcium or H2O . After 20min , samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was supplemented with Myc - XErp1 wt IVT at the indicated dilutions . An empty IVT reaction not expressing Myc - XErp1 served as control . All reactions were divided and treated with DMSO or CsA . Calcium was added to all reactions and samples were taken at the indicated time points , treated with λ - phosphatase and immunoblotted for XErp1 , the Myc - tag , cyclin B2 and α - tubulin . Asterisks indicate unspecific bands . CSF extract was supplemented with Myc - XErp1 wt IVT . The reaction was divided and treated with DMSO or CsA . Both reactions were treated with calcium , samples were taken at the indicated time points and as indicated incubated with λ - phosphatase . Samples were immunoblotted for XErp1 and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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Figure 1CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


Figure 2CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT and as indicated treated with DMSO or CsA . Both reactions were treated with calcium and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag , cyclin B2 and α - tubulin . A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A . Arrow marks the meiotic phosphorylation state of Myc - XErp1 . Asterisk indicates unspecific bands . CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT that was either wild - type or mutated to alanine at the p90RSK - target sites S335 , T336 and S342 ( Rsk3A ) . Both reactions were divided and treated with DMSO or CsA . Calcium was added and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands . CSF extract was treated with Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT . An empty IVT reaction not expressing XErp1 was used as control . The extracts were treated with DMSO or CsA as indicated . Calcium was added , samples were taken at the indicated time points and immunoblotted for cyclin B2 , pSer335 XErp1 and α - tubulin . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisks indicate unspecific bands . Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT was in vitro phosphorylated by recombinant PKA and isolated by α - Myc immunoprecipitation . The immunoprecipitate was supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1 . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisk indicates the IgG heavy chain .

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Figure 2CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.


Figure 3CSF extract was supplemented with mRNA encoding Flag - Cdc20 that was either wild - type or mutated to serine at Thr64 , Thr68 and Thr79 ( 3TS ) . After expression , calcium was added , samples were taken at the indicated time points and resolved by either conventional or Phos - tagTM SDS - PAGE . Samples were immunoblotted for the Flag - tag , Cdc27 and cyclin B2 . CSF extract was supplemented with Myc - XErp1 wt IVT and treated with DMSO or CsA . Calcium was added , samples were taken at the indicated time points , resolved by either conventional or Phos - tagTM SDS - PAGE and immunoblotted for Cdc20 , Cdc27 and cyclin B2 . A control sample was treated with λ - phosphatase . A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig . EV4A . The cyclin B2 membrane was stripped and reprobed for α - tubulin . CSF extract was supplemented with Flag - Cdc20 wt IVT coupled to α - Flag beads . Calcium was added and Flag - Cdc20 was isolated by α - Flag immunoprecipitation at the indicated time points . Input and pellet ( IP : Flag ) samples were immunoblotted for the Flag - tag , pThr68 Cdc20 and p150 ( Glued ) . The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2 . Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain . Asterisks in other immunoblots indicate unspecific bands . CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the Flag - tag and pThr68 Cdc20 . The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA . Asterisk indicates the IgG heavy chain .

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Figure 3CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.


figf1 ( a ) Left , cells were fixed and stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . Solid arrows indicate cells with active Src in adhesions ; dashed arrows show cells with active Src in puncta . Scale bars , 20 μm . Right , percentage of cells with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . ( b ) Cells were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Merged and higher - magnification images of the outlined areas are also shown . Solid and dashed arrows show Src localization to adhesions or to puncta , respectively . Scale bars , 20 μm . ( c ) Lysates from cells were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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figf1(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3).(b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm.(c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf2 ( a ) Cells were fixed and stained for anti - pTyr - 416 - Src ( red ) and co - stained for anti - LC3B , anti - Atg7 or anti - Atg12 ( all green ) . Scale bars , 20 μm . ( b ) Higher - magnification images of the areas outlined in a . Solid arrows indicate co - localization in focal adhesions and dashed arrows show puncta in the cytoplasm . ( c ) LC3B was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - LC3B antibodies . ( d ) Cells were transfected with 40 nM scrambled , Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti - Atg5 , anti - Atg12 and anti - actin antibodies ( lower panels ) or fixed and stained for anti - pTyr - 416 - Src ( green ) , anti - paxillin ( red ) and with DAPI ( blue ) . Dashed arrows point to intracellular phospho - Src - containing puncta ; solid arrows point to peripheral phospho - Src . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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figf2(a) Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. (b) Higher-magnification images of the areas outlined in a. Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm.(c) LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies.(d) Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


figf3 ( a ) Top , cells were transiently transfected with an RFP - GFP tandem fluorescent - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , the number of yellow puncta and the number of RFP LC3 - positive puncta in the merged images were counted and the total number of puncta per cell was calculated . Data are presented as mean ± s . d . ( n = 3 ) . b ) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti - pTyr - 416 - Src , anti - Src , anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . ( c ) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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figf3(a) Top, cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3).b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf4 ( a ) Top , FAK − / − cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti - Src antibody ( green ) . Solid arrows indicate Src at the cell periphery ; dashed arrows indicate Src in autophagosomes . Scale bars , 20 μm . Bottom , quantification of dasatinib - treated cells with Src in puncta . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . Middle , lysates from cells treated with dasatinib were immunoblotted with anti - Src and anti - pTyr - 416 - Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . ( b ) Top , FAK − / − cells were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) and stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , quantification of cells with co - localization between Src and LC3B . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . ( c ) Left , FAK − / − cells stably re - expressing wild - type FAK , FAKY397F or FAKY4F - Y9F were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate active Src in autophagosomes . Scale bars , 20 μm . Right , lysates from these cells were also immunoblotted with anti - FAK and anti - actin antibodies . ( d ) Left , wild - type FAK , FAKY397F or FAKY4F - Y9F cells were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization and dotted arrows indicate its absence . Scale bars , 20 μm . ( e ) Lysates were immunoblotted with anti - LC3B and actin antibodies in the absence and presence of chloroquine . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

figf4(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.(b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3).(c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies.(d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm.(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.


figf5 ( a ) Wild - type FAK cells were suspended in 1 . 4 % methylcellulose solution in growth media and plated on agarose for 3 days . Top and bottom right , cells were recovered and cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) and with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) and with DAPI ( blue ) . Higher - magnification images of the outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive puncta . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 and anti - Src antibodies . ( b ) FAK + / + cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . A higher - magnification image of the area outlined is also shown . Scale bar , 20 μm . ( c ) LC3B was immunoprecipitated ( IP ) from adherent or suspended FAK + / + and then immunoblotted with anti - Src and anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

figf5(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.(c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.