Figure 3A . Immunofluorescence of collagen matrix sections co - stained for cleaved collagen neo - epitope Col1 3 / 4C and DAPI ( left panels ) . Second harmonic generation ( SHG ) images of collagen fibers within 3 - dimensional collagen matrices at 15 µm and 45 µm below the cell surface ( middle panels ) . Scale bar = 20 µm . Graphs indicate the mean ± SEM fibrillary collagen areas at 2 . 5 µm increments below the cell / collagen interface ( n = 5 ) . SHG fibrillar collagen ( red ) ; cells ( green ) . B , C . Grey level correlation matrix ( GLCM ) texture analysis of SHG correlation ( n = 6 ) and contrast ( n = 5 ) . Mean ± SEM at each distance . D . Transmission electron microscopy of a collagen matrix section . Scale bar = 1 µm . Positions of magnified areas indicated by corresponding colored squares . E . Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid . Yellow arrows indicate long collagen bundles , Red arrows indicate short collagen bundles . Scale bar = 0 . 5 µm .

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

Figure 3A. Immunofluorescence of collagen matrix sections co-stained for cleaved collagen neo-epitope Col1 3/4C and DAPI (left panels). Second harmonic generation (SHG) images of collagen fibers within 3-dimensional collagen matrices at 15 µm and 45 µm below the cell surface (middle panels). Scale bar = 20 µm. Graphs indicate the mean ± SEM fibrillary collagen areas at 2.5 µm increments below the cell/collagen interface (n=5). SHG fibrillar collagen (red); cells (green).B, C. Grey level correlation matrix (GLCM) texture analysis of SHG correlation (n=6) and contrast (n=5). Mean ± SEM at each distance.D. Transmission electron microscopy of a collagen matrix section. Scale bar = 1 µm. Positions of magnified areas indicated by corresponding colored squares.E. Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid. Yellow arrows indicate long collagen bundles, Red arrows indicate short collagen bundles. Scale bar = 0.5 µm.


Figure 4A . Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate ( FDR ) < 5 % , with log10 fold - change ( FC ) in expression in 4HT treated ROCK1 : ER expressing cells relative to GFP : ER expressing cells ( left ) or 4HT treated ROCK2 : ER expressing cells relative to GFP : ER expressing cells ( right ) versus log10 adjusted p value ( n = 3 ) . ROCK1 : ER + 4HT vs GFP : ER + 4HT ; 3828 genes . ROCK2 : ER + 4HT vs GFP : ER + 4HT ; 4481 genes . C . Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion : Cell - matrix interaction . High relative expression in red and low relative expression in blue . D . MMP average RNA sequence reads . Mean ± SEM ( n = 3 ) * adjusted p - value < 0 . 05 . E . Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± SEM ( n = 4 ) , p value by unpaired t test . Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . F . Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± SEM ( n = 4 ) , p value by unpaired t test . Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . G . MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma . Scale bar = 100 µm ( left ) . Histoscores of MMP10 staining ( right ) in normal pancreas ( n = 5 ) , and pancreas adenocarcinoma grade 1 ( n = 23 ) , grade 2 ( n = 27 ) and grade 3 ( n = 13 ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

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

Figure 4A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes.C. Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion: Cell-matrix interaction. High relative expression in red and low relative expression in blue.D. MMP average RNA sequence reads. Mean ± SEM (n=3) * adjusted p-value <0.05.E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.G. MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma. Scale bar = 100 µm (left). Histoscores of MMP10 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma grade 1 (n=23), grade 2 (n=27) and grade 3 (n=13). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


Figure 5A . Confocal microscope images of ROCK1 : ER - expressing cells co - stained for F - actin , MMP10 ( left ) or MMP13 ( right ) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h . Multiple z - planes were used to generate x - z and y - z images . Scale bar = 5 µm . B . Representative immunoblot of MMP13 and MMP10 in conditioned media , as well as MMP10 and GAPDH in cell lysates . Cells were treated with vehicle ( - ) or 1 µM 4HT for 48 h . C . Representative stained gel of ultracentrifuge - enriched microvesicle protein from GFP : ER or ROCK1 : ER - expressing cell - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h ( top ) . Absolute arbitrary unit values for total microvesicle protein levels in stained gels ( bottom ) . Means ± SEM ( n = 4 ) , p value by ratio paired t - test . D . Transmission electron microscopy of ultracentrifuge - enriched microvesicles ( top center ) , and immunogold labeling of MMP10 in ROCK1 : ER 4HT - treated microvesicles , indicated by red arrows ( top right ) . D . Representative immunoblot of MMP10 and caveolin in ultracentrifuge - enriched microvesicle proteins from cell - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h as well as MMP10 , caveolin and GAPDH in cell lysates ( top left ) . Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation - enriched microvesicle proteins ( bottom left ) . Means ± SEM ( n = 4 ) , p value by ratio paired t test . Ratios of MMP10 to caveolin ( bottom center ) or to total microvesicle proteins ( bottom right ) . Means ± SEM ( n = 4 ) , p value by ratio paired t test . E . Absolute arbitrary unit values for caveolin levels in western blots of ultracentrifugation - enriched microvesicle proteins . Means ± SEM ( n = 4 ) , p value by ratio paired t test . Ratio of caveolin to total microvesicle proteins . Means ± SEM ( n = 4 ) , p value by ratio paired t test . F . Ratios of MMP10 to GAPDH and caveolin to GAPDH in western blots of whole cell lysates . Means ± SEM ( n = 3 ) , p value by ratio paired t test .

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

Figure 5A. Confocal microscope images of ROCK1:ER-expressing cells co-stained for F-actin, MMP10 (left) or MMP13 (right) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h. Multiple z-planes were used to generate x-z and y-z images. Scale bar = 5 µm.B. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (-) or 1 µM 4HT for 48 h.C. Representative stained gel of ultracentrifuge-enriched microvesicle protein from GFP:ER or ROCK1:ER-expressing cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM (n=4), p value by ratio paired t-test.D. Transmission electron microscopy of ultracentrifuge-enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT-treated microvesicles, indicated by red arrows (top right).D. Representative immunoblot of MMP10 and caveolin in ultracentrifuge-enriched microvesicle proteins from cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h as well as MMP10, caveolin and GAPDH in cell lysates (top left). Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation-enriched microvesicle proteins (bottom left). Means ± SEM (n=4), p value by ratio paired t test. Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM (n=4), p value by ratio paired t test.E. Absolute arbitrary unit values for caveolin levels in western blots of ultracentrifugation-enriched microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test. Ratio of caveolin to total microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test.F. Ratios of MMP10 to GAPDH and caveolin to GAPDH in western blots of whole cell lysates. Means ± SEM (n=3), p value by ratio paired t test.


Figure 6A . Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1 - FITC ( Col1 - FITC ) . Co - staining for F - actin , MMP10 ( red ) and DAPI ( blue ) . Scale bar = 10 µm . B . Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h . Means ± SEM ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . C . Quantification of collagen degradation by cells treated with 1 µM 4HT , 4HT + DMSO vehicle , 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h . Means ± SEM ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . D . Immunofluorescence of collagen matrix sections co - stained for MMP10 and DAPI . Scale bar = 20 µm . E . Mmp13 immunohistochemistry stained sections of collagen matrices . Scale bar = 50 µm .

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

Figure 6A. Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1-FITC (Col1-FITC). Co-staining for F-actin, MMP10 (red) and DAPI (blue). Scale bar = 10 µm.B. Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.C. Quantification of collagen degradation by cells treated with 1 µM 4HT, 4HT + DMSO vehicle, 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.D. Immunofluorescence of collagen matrix sections co-stained for MMP10 and DAPI. Scale bar = 20 µm.E. Mmp13 immunohistochemistry stained sections of collagen matrices. Scale bar = 50 µm.


Figure 7A . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days , in the absence ( top ) or presence of 10 µM GM6001 ( bottom ) . Scale bar = 100 µm . B . Invasion index of KPflC cells . Means ± SEM ( n = 4 ) , p value by unpaired t test . C . Quantification of cell number at the collagen matrix surface per 0 . 046 mm2 field . Means ± SEM ( n = 20 ) , p value by unpaired t test . D . Cell proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . E . Ki67 positive cell percentages at the collagen matrix surface . Means ± SEM ( n = 20 ; n = 19 for GFP : ER / vehicle , n = 18 for GFP / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . F . Ki67 positive cell percentages in collagen matrix . Means ± SEM ( n = 20 ; n = 8 for GFP : ER / vehicle , n = 10 for GFP : ER / GM6001 , n = 17 for ROCK1 : ER / GM6001 , n = 15 for ROCK2 : ER / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . G . The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001 . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . H . The increase in viable cell numbers of cells plated on collagen1 ( Col1 ) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001 . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

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

Figure 7A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 µM GM6001 (bottom). Scale bar = 100 µm.B. Invasion index of KPflC cells. Means ± SEM (n=4), p value by unpaired t test.C. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=20), p value by unpaired t test.D. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.E. Ki67 positive cell percentages at the collagen matrix surface. Means ± SEM (n=20; n=19 for GFP:ER/vehicle, n=18 for GFP/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Ki67 positive cell percentages in collagen matrix. Means ± SEM (n=20; n=8 for GFP:ER/ vehicle, n=10 for GFP:ER/GM6001, n=17 for ROCK1:ER/GM6001, n=15 for ROCK2:ER/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.G. The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. The increase in viable cell numbers of cells plated on collagen1 (Col1) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


Figure 8A . Survival analysis of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; ( KPC ) mice without ( n = 13 ) or with Fasudil treatment ( n = 13 ) . Survival p - value determined by Gehan - Breslow - Wilcoxon test . B . Representative images of picrosirius red stained endpoint tumors from KPC mice . Scale bar = 1 mm . C . Picrosirius red percentage positive staining area ( top ) or average staining intensity of positive area ( bottom ) in pancreatic tumors from vehicle ( n = 8 ) and Fasudil ( n = 7 ) treated mice . Box plots with exact p value by Mann - Whitney test .

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

Figure 8A. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; (KPC) mice without (n=13) or with Fasudil treatment (n=13). Survival p-value determined by Gehan-Breslow-Wilcoxon test.B. Representative images of picrosirius red stained endpoint tumors from KPC mice. Scale bar = 1 mm.C. Picrosirius red percentage positive staining area (top) or average staining intensity of positive area (bottom) in pancreatic tumors from vehicle (n=8) and Fasudil (n=7) treated mice. Box plots with exact p value by Mann-Whitney test.


Figure 1B . Binding assay using MBP - fused Apc1 - loop500 fragments . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , 2A ( T532A / T539A ) and S558A ) was incubated with anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining . D . Specific binding of Plx1 - PBD to Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives 2A ( T532A / T539A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated in the presence of 10 µg of WT Plx1 - PBD for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by

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

Figure 1B. Binding assay using MBP-fused Apc1-loop500 fragments. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.D. Specific binding of Plx1-PBD to Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of 10 µg of WT Plx1-PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie


Figure 2A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant WT or Apc - loop500 mutant APC / Cs with mutations ( 1 - T532A , 1 - T539A , 1 - T532A / T539A ( 1 - 2A ) and 1 - S558A ) was incubated with APC / C depleted ( ΔAPC ) interphase extract ( Inter ) or ΔAPC anaphase extracts supplemented with non - degradable cyclin B ( Ana ) at 23 ˚ C for 1 h . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . E . In vitro phosphorylation by Plx1 . Apc3 - loop fragment , α - Casein , N - terminus of Cdc20 ( N159 ) or MBP protein was incubated with purified Plx1 in the presence of [ γ32P ] - ATP at 23 ˚ C for indicated times , separated by SDS - PAGE and detected by autoradiography and Coomassie brilliant blue ( CBB ) staining .

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

Figure 2A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant WT or Apc-loop500 mutant APC/Cs with mutations (1-T532A, 1-T539A, 1-T532A/T539A (1-2A) and 1-S558A) was incubated with APC/C depleted (ΔAPC) interphase extract (Inter) or ΔAPC anaphase extracts supplemented with non-degradable cyclin B (Ana) at 23˚C for 1 h. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies.E. In vitro phosphorylation by Plx1. Apc3-loop fragment, α-Casein, N-terminus of Cdc20 (N159) or MBP protein was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography and Coomassie brilliant blue (CBB) staining.


Figure 3C . Plx1 - binding assay using MBP - fused Apc1 - loop500 fragments with mutations in B56 - binding site . The Plx1 - binding to Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was analysed as described in Fig 1B . D . Quantification of ( C ) . The bar graph is quantification of bound Plx1 . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments . E . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . MBP - fused Apc1 - loop500 WT or its derivatives ( T532A , T539A , T532A / T539A ( 2A ) and S558A ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining . The recovery of WT ( x1 / 2 and x1 ) was run as a standard . F . Quantification of ( E ) . The bar graph is quantification of bound B56γ . The intensities of WT were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3C. Plx1-binding assay using MBP-fused Apc1-loop500 fragments with mutations in B56-binding site. The Plx1-binding to Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was analysed as described in Fig 1B. D. Quantification of (C). The bar graph is quantification of bound Plx1. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard. F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.


Figure 4A . Plx1 and PP2A - B56 competes for Apc1 - loop500 . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with the 35S - labelled Flag - B56γ in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with indicated amounts of WT Plx1 - PBD or Plx1 - PBD Pincer mutant ( Pin ) for 15 min . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or Coomassie brilliant blue ( CBB ) staining . C . Dephosphorylation of Apc1 - loop500 by PP2A - B56 in the presence of Plx1 - PBD . MBP - fused Apc1 - loop500 WT or its derivatives ( L557A / V560A or E562A ) was incubated in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1 h , and further incubated with 10 µg of WT Plx1 - PBD for 15 min . The complexes were isolated by amylose beads and incubated in the presence ( 78 nM ) or absence of purified PP2A - B56γ at 23 ˚ C for 20 min . The proteins were recovered by amylose beads , and analysed as described in ( B ) .

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

Figure 4A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining.C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).


Figure 5A . Phosphorylation kinetics of T532 , T539 and S558 on Apc1 - loop500 in anaphase extracts . MBP - fused Apc1 - loop500 WT or its derivatives ( ∆ 11 , deletion of 11 residues including the B56 - binding motif ) was incubated with Xenopus egg extracts in the presence of non - degradable cyclin B at 23 ˚ C . The samples taken as indicated were analysed by SDS - PAGE and immunoblotting using phospho - specific antibodies ( pT532 , pT539 and pS558 ) or MBP antibody . To monitor CDK activity , Apc3 antibody was used ( bottom ) . The signals of a phospho - specific antibody and MBP antibody on the same membrane were detected in the 800 - and 700 - nm channels , respectively . The two channels were pseudo - coloured ( green ; pT532 , pT539 and pS558 , red ; MBP ) and overlaid in the bottom panel ( Overlay ) . The signal of pS558 was not detected in ∆ 11 mutant because ∆ 11 mutation includes S558 .

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

Figure 5A. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 in anaphase extracts. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with Xenopus egg extracts in the presence of non-degradable cyclin B at 23˚C. The samples taken as indicated were analysed by SDS-PAGE and immunoblotting using phospho-specific antibodies (pT532, pT539 and pS558) or MBP antibody. To monitor CDK activity, Apc3 antibody was used (bottom). The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was not detected in ∆11 mutant because ∆11 mutation includes S558.


Figure 6A . Cdc20 binding assay in Xenopus egg extracts . The Cdc20 - binding activities of WT APC / C and Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) were analysed as described in Fig 2A . B . Quantification of ( A ) . The bar graph is quantification of bound Cdc20 . The intensities of WT control at Ana - 45 min were arbitrarily set to 1 . 0 . Error bars , SEM from three independent experiments . D . APC / C dephosphorylation after inactivation of CDK . The purified recombinant WT or Apc - loop500 - mutant APC / Cs with mutations ( 1 - L557A / V560A or 1 - E562A ) was incubated with APC / C depleted ( ΔAPC ) in anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 1h ( Ana ) . After addition of p27 ( 0 . 3 µM ) , the samples taken at time points shown were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ' Inter ' denotes interphase .

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Figure 6A. Cdc20 binding assay in Xenopus egg extracts. The Cdc20-binding activities of WT APC/C and Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) were analysed as described in Fig 2A. B. Quantification of (A). The bar graph is quantification of bound Cdc20.The intensities of WT control at Ana-45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. D. APC/C dephosphorylation after inactivation of CDK. The purified recombinant WT or Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) was incubated with APC/C depleted (ΔAPC) in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1h (Ana). After addition of p27 (0.3 µM), the samples taken at time points shown were analysed by SDS-PAGE and immunoblotting with indicated antibodies. 'Inter' denotes interphase.


Figure 7A . The mutations in CDK sites on Apc3 - loop reduces phosphorylation of T532 on Apc1 . The purified recombinant WT or a mutant APC / C with mutations at nine CDK sites in Apc3 - loop ( 3 - 9A ) was incubated with APC / C - depleted ( ΔAPC ) anaphase extracts supplemented with non - degradable cyclin B at 23 ˚ C for 60 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads , and the phosphorylation status of T532 in Apc1 was analysed by SDS - PAGE and immunoblotting with phospho - specific ( pT532 ) or Apc1 ( pan - Apc1 ) antibodies .

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Figure 7A. The mutations in CDK sites on Apc3-loop reduces phosphorylation of T532 on Apc1. The purified recombinant WT or a mutant APC/C with mutations at nine CDK sites in Apc3-loop (3-9A) was incubated with APC/C-depleted (ΔAPC) anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 60 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the phosphorylation status of T532 in Apc1 was analysed by SDS-PAGE and immunoblotting with phospho-specific (pT532) or Apc1 (pan-Apc1) antibodies.


Figure 1B Phos ‐ tag gel following complete conversion of Ub to phosphoUb , using GST ‐ PhPINK1 . A Ser65 mutant Ub is not modified . C ESI ‐ MS analysis of pure Ub ( left , mass 8 , 565 . 65 Da , reference 8 , 565 . 80 Da ) and purified phosphoUb ( right , mass 8 , 645 . 61 Da , reference 8 , 645 . 80 Da ) . The difference of 80 Da corresponds to one phosphate group . D AQUA analysis of PhPINK1 ‐ phosphorylated Lys63 ‐ linked hexaUb . The Lys63 peptide ( K63 ) is modified by Ser65 phosphorylation during the time course , as observed by rise of the Lys63 / pSer65 peptide ( K63pS65 ) . E AQUA analysis after 180 min as in ( D ) with Lys6 ‐ , Lys11 ‐ , Lys33 ‐ , Lys48 ‐ , Lys63 ‐ , and Met1 ‐ linked tetraUb ( K6 , K11 , K33 , K48 , K63 , M1 ) . For all tetraUb chains , Ser65 is phosphorylated to greater than 80 % after incubation with PhPINK1 for 180 min . F Time course analysis of autoubiquitination reactions with unphosphorylated full ‐ length humanParkin ( hParkin ) and Ub ( left ) , a 80 % Ub / 20 % phosphoUb mixture ( middle ) and 100 % phosphoUb ( right ) . G Analysis of different ratios of Ub : phosphoUb in a 120 ‐ min ubiquitination reaction of full ‐ length hParkin . Parkin is activated by a mixture of Ub and phosphoUb , and is much less active in a reaction with no phosphoUb as well as with 100 % phosphoUb .

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Figure 1B Phos‐tag gel following complete conversion of Ub to phosphoUb, using GST‐PhPINK1. A Ser65 mutant Ub is not modified.C ESI‐MS analysis of pure Ub (left, mass 8,565.65 Da, reference 8,565.80 Da) and purified phosphoUb (right, mass 8,645.61 Da, reference 8,645.80 Da). The difference of 80 Da corresponds to one phosphate group.D AQUA analysis of PhPINK1‐phosphorylated Lys63‐linked hexaUb. The Lys63 peptide (K63) is modified by Ser65 phosphorylation during the time course, as observed by rise of the Lys63/pSer65 peptide (K63pS65).E AQUA analysis after 180 min as in (D) with Lys6‐, Lys11‐, Lys33‐, Lys48‐, Lys63‐, and Met1‐linked tetraUb (K6, K11, K33, K48, K63, M1). For all tetraUb chains, Ser65 is phosphorylated to greater than 80% after incubation with PhPINK1 for 180 min.F Time course analysis of autoubiquitination reactions with unphosphorylated full‐length humanParkin (hParkin) and Ub (left), a 80% Ub/20% phosphoUb mixture (middle) and 100% phosphoUb (right).G Analysis of different ratios of Ub:phosphoUb in a 120‐min ubiquitination reaction of full‐length hParkin. Parkin is activated by a mixture of Ub and phosphoUb, and is much less active in a reaction with no phosphoUb as well as with 100% phosphoUb.


Figure 2A 1H , 15N ‐ BEST ‐ TROSY spectrum of phosphoUb ( shades of blue ) overlaid with wild ‐ type ( wt ) Ub ( orange ) . The phosphoUb spectrum contains 130 non ‐ sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B - D . B - D Weighted chemical shift perturbation ( CSP ) graphs for ( B ) wt Ub versus major phosphoUb species , ( C ) wt Ub versus minor phosphoUb species , ( D ) major versus minor phosphoUb species .

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Figure 2A 1H, 15N‐BEST‐TROSY spectrum of phosphoUb (shades of blue) overlaid with wild‐type (wt) Ub (orange). The phosphoUb spectrum contains 130 non‐sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B-D.B-D Weighted chemical shift perturbation (CSP) graphs for (B) wt Ub versus major phosphoUb species, (C) wt Ub versus minor phosphoUb species, (D) major versus minor phosphoUb species.


Figure 3A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit ( Table ) . Ub molecules are colored differently , and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms . B Six of eight phosphoUb molecules form similar dimer interactions , where phosphoUb molecules interact via their Ile44 patch . This resembles the crystal structure of Lys48 ‐ linked diUb [ pdb ‐ id 1aar ( Cook et al , ) ] . Atoms for pSer65 are shown as in ( A ) , and the Ile44 side chain is shown as blue sticks . C Representative 2 | Fo | ‐ | Fc | electron density , contoured at 1 σ , covering the phosphorylation site . pSer65 is shown in ball ‐ and ‐ stick representation , and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms . D In wt Ub ( left ) , Ser65 forms a hydrogen bond with the backbone amide of Gln62 . Upon phosphorylation ( right ) , an oxygen atom from the phosphate forms the same backbone hydrogen bond . E Cartoon representation and electrostatic surface potential of Ub ( left ) and phosphoUb ( right ) in identical orientations . Electrostatics were calculated using CHARMM ( www . charmm - gui . org ) and are colored from red ( negative potential ) to blue ( positive potential ) .

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Figure 3A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms.B Six of eight phosphoUb molecules form similar dimer interactions, where phosphoUb molecules interact via their Ile44 patch. This resembles the crystal structure of Lys48‐linked diUb [pdb‐id 1aar (Cook et al, )]. Atoms for pSer65 are shown as in (A), and the Ile44 side chain is shown as blue sticks.C Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms.D In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond.E Cartoon representation and electrostatic surface potential of Ub (left) and phosphoUb (right) in identical orientations. Electrostatics were calculated using CHARMM (www.charmm-gui.org) and are colored from red (negative potential) to blue (positive potential).


Figure 4A Long ‐ range HNCO NMR experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species . Through H bond correlations ( blue ) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum ( red ) . In the major form of phosphoUb ( left ) , the HN of Ile44 is a H bond donor to His68 , as seen in wt Ub . In the minor form of phosphoUb ( right ) , the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70 . C Structure of the major form of phosphoUb , where phosphoUb residues 1 - 61 are shown under a surface , and the β5 strand is shown in stick representation with red oxygen , blue nitrogen and purple phosphorus atoms . The four core Leu residues are shown in green . D Model of the phosphoUb minor species as in ( C ) in which the β5 strand is retracted into the protein core by two residues . This extends the β4 - β5 loop and significantly shortens the C ‐ terminal tail of Ub .

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Figure 4A Long‐range HNCO NMR experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species. Through H bond correlations (blue) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum (red). In the major form of phosphoUb (left), the HN of Ile44 is a H bond donor to His68, as seen in wt Ub. In the minor form of phosphoUb (right), the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70.C Structure of the major form of phosphoUb, where phosphoUb residues 1-61 are shown under a surface, and the β5 strand is shown in stick representation with red oxygen, blue nitrogen and purple phosphorus atoms. The four core Leu residues are shown in green.D Model of the phosphoUb minor species as in (C) in which the β5 strand is retracted into the protein core by two residues. This extends the β4-β5 loop and significantly shortens the C‐terminal tail of Ub.


Figure 5A E1 ‐ mediated charging of E2 enzymes by Ub and phosphoUb in a time course analysis . Non ‐ reducing Coomassie ‐ stained 4 - 12 % SDS - PAGE gradient gels are shown , and bands are labeled . " ˜ Ub " refers to generation of a thioester , while " Ub ‐ " refers to additional covalent ubiquitination of the E2 enzyme . * contaminant from E2 purification . B SAXS ‐ derived ensemble of UBE2D3 ( Pruneda et al , ) , where the E2 is shown under a blue surface and the Ub is shown as a green ribbon . Ser65 is shown in sphere representation . Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme . PDB files were downloaded from the Klevit ‐ lab website ( http : / / depts . washington . edu / klvtlab / ) . C Quantitative mass spectrometry analysis for UBE2T , UBE2E1 and UBE2R1 with Ub and phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb ( see also Supplementary Fig S10 ) . D Charging of UBE2N , and UBE2N / UBE2V1 ‐ mediated Lys63 chain assembly with Ub and phosphoUb . Charging of UBE2N as in ( A ) proceeds identically with both Ub and phosphoUb ( left 6 lanes ) , while UBE2V1 ‐ mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb ( right 6 lanes and Western blot ) . E Structure of Saccharomyces cerevisiae Ubc13 ‐ Mms2 ( blue / red , Mms2 is a homolog of UBE2V1 ) with the acceptor Ub ( cyan ) bound non ‐ covalently to Mms2 [ pdb ‐ id 2gmi ( Eddins et al , ) ] . Ser65 of the acceptor Ub interacts with Mms2 , and Ser65 phosphorylation would prevent binding due to Mms2 Glu18 . F PhosphoUb competition assays for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation . Time course with K63R Ub ( donor ) and Ub ΔGG ( acceptor ) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb , revealing phosphoUb does not compete with Ub for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation .

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Figure 5A E1‐mediated charging of E2 enzymes by Ub and phosphoUb in a time course analysis. Non‐reducing Coomassie‐stained 4-12% SDS-PAGE gradient gels are shown, and bands are labeled. "˜Ub" refers to generation of a thioester, while "Ub‐"refers to additional covalent ubiquitination of the E2 enzyme. * contaminant from E2 purification.B SAXS‐derived ensemble of UBE2D3 (Pruneda et al, ), where the E2 is shown under a blue surface and the Ub is shown as a green ribbon. Ser65 is shown in sphere representation. Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme. PDB files were downloaded from the Klevit‐lab website (http://depts.washington.edu/klvtlab/).C Quantitative mass spectrometry analysis for UBE2T, UBE2E1 and UBE2R1 with Ub and phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb (see also Supplementary Fig S10).D Charging of UBE2N, and UBE2N/UBE2V1‐mediated Lys63 chain assembly with Ub and phosphoUb. Charging of UBE2N as in (A) proceeds identically with both Ub and phosphoUb (left 6 lanes), while UBE2V1‐mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb (right 6 lanes and Western blot).E Structure of Saccharomyces cerevisiae Ubc13‐Mms2 (blue/red, Mms2 is a homolog of UBE2V1) with the acceptor Ub (cyan) bound non‐covalently to Mms2 [pdb‐id 2gmi (Eddins et al, )]. Ser65 of the acceptor Ub interacts with Mms2, and Ser65 phosphorylation would prevent binding due to Mms2 Glu18.F PhosphoUb competition assays for UBE2N/UBE2V1‐mediated Lys63 diUb formation. Time course with K63R Ub (donor) and Ub ΔGG (acceptor) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb, revealing phosphoUb does not compete with Ub for UBE2N/UBE2V1‐mediated Lys63 diUb formation.


Figure 6A Time course for a ligase reaction with GST ‐ tagged cIAP1 ( aa 363 - 614 ) , UBE2D1 and Ub or phosphoUb . See Supplementary Fig S11D for a reaction with UBE2D3 . B Anti ‐ Ub Western blot of an identical reaction run for 3 h . C Comparison of the number of Ub chain linkages in the reaction in ( B ) . D Ub chain linkage profile for the reaction in ( B ) . E Reaction of GST ‐ tagged TRAF6 ( aa 1 - 285 ) with UBE2D3 as in ( A ) . See Supplementary Fig S11E and F for additional TRAF6 reactions . F Anti ‐ Ub Western blot of GST ‐ TRAF6 / UBE2D1 run for 3 h . G Comparison of the number of Ub chain linkages in the reaction in ( F ) . H Linkage composition in GST ‐ TRAF6 ‐ UBE2D1 reactions reveals 58 % Lys63 chains with wt Ub and UBE2D1 . Significantly smaller quantities of linkages are assembled with phosphoUb . I - K Chain assembly as in ( A ) with UBE2L3 and ( I ) GST ‐ tagged NEDD4L ( aa 576 - 955 ) ( Mund Pelham , ) , ( J ) NleL ( aa 170 - 782 ) ( Hospenthal et al , ) and ( K ) HOIP RBR ‐ LDD ( aa 699 - 1072 ) ( Smit et al , ) . While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb , HOIP is significantly less active with phosphoUb . See also Supplementary Fig S11G and H .

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Figure 6A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363-614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3.B Anti‐Ub Western blot of an identical reaction run for 3 h.C Comparison of the number of Ub chain linkages in the reaction in (B).D Ub chain linkage profile for the reaction in (B).E Reaction of GST‐tagged TRAF6 (aa 1-285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions.F Anti‐Ub Western blot of GST‐TRAF6/UBE2D1 run for 3 h.G Comparison of the number of Ub chain linkages in the reaction in (F).H Linkage composition in GST‐TRAF6‐UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb.I-K Chain assembly as in (A) with UBE2L3 and (I) GST‐tagged NEDD4L (aa 576-955) (Mund Pelham, ), (J) NleL (aa 170-782) (Hospenthal et al, ) and (K) HOIP RBR‐LDD (aa 699-1072) (Smit et al, ). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.


Figure 7A Structure of TAB2 NZF ( red ) in complex with Lys63 diUb ( cyan ) [ pdb ‐ id 2wwz ( Kulathu et al , ) ] . Ser65 for both Ub moieties is shown as spheres and highlighted with a blue circle . B Pull ‐ down assays with the Lys63 ‐ specific TAB2 NZF against different tetraUb ( Ub4 ) species are shown . Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF . Met1 ‐ linked tetraUb does not interact with TAB2 NZF , independent of Ser65 phosphorylation .

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Figure 7A Structure of TAB2 NZF (red) in complex with Lys63 diUb (cyan) [pdb‐id 2wwz (Kulathu et al, )]. Ser65 for both Ub moieties is shown as spheres and highlighted with a blue circle.B Pull‐down assays with the Lys63‐specific TAB2 NZF against different tetraUb (Ub4) species are shown. Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF. Met1‐linked tetraUb does not interact with TAB2 NZF, independent of Ser65 phosphorylation.


Figure 8A Structure of USP2 ( red ) bound to Ub ( cyan ) [ pdb ‐ id 2hd5 ( Renatus et al , ) ] , with a close ‐ up on Ub Ser65 in the distal Ub . A negative charge at the USP2 Asp345 position is conserved in most USP enzymes ( Ye et al , ) . B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) analysis . Silver stained time course gels are shown . All DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with the exception of vOTU and USP21 . B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) analysis . Silver ‐ stained time ‐ course gels are shown . All DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with the exception of vOTU and USP21 . I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs . Cleavage assays against the diUb panel reveals preference for Lys6 ‐ linked chains . J USP30 activity is decreased against phosphorylated Lys6 diUb . K - N Deubiquitinase assays against phosphorylated tetraUb . The activity of AMSH ( K ) , TRABID ( L ) , OTUB1 ( M ) and OTULIN ( N ) are inhibited with Lys63 , Lys33 , Lys48 and Met1 phosphorylated tetraUb , respectively .

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Figure 8A Structure of USP2 (red) bound to Ub (cyan) [pdb‐id 2hd5 (Renatus et al, )], with a close‐up on Ub Ser65 in the distal Ub. A negative charge at the USP2 Asp345 position is conserved in most USP enzymes (Ye et al, ).B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver stained time course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver‐stained time‐course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs. Cleavage assays against the diUb panel reveals preference for Lys6‐linked chains.J USP30 activity is decreased against phosphorylated Lys6 diUb.K-N Deubiquitinase assays against phosphorylated tetraUb. The activity of AMSH (K), TRABID (L), OTUB1 (M) and OTULIN (N) are inhibited with Lys63, Lys33, Lys48 and Met1 phosphorylated tetraUb, respectively.


Figure 1Dose - response curve of HF and DWN12088 in WI - 26 VA4 cells . The IC50 value for collagen level was measured as a representative of chemical efficacy . The cells were starved of serum for 6 hr and then incubated with the indicated concentrations of compounds in the presence of TGF - β ( 2 ng / ml ) for 72 hr . The secreted pro - collagen I levels were determined by ELISA . The CC50 values were determined by CellTiter - Glo assay . Dose - response curves were determined by combining at least 2 biologically independent experiments . The IC50 values were calculated using GraphPad Prism 7 . 0 ( Collagen level , n = 5 from three independent experiments ( singlicate for one experiment and duplicate for two experiments ; mean ± SEM ) ; Celltiter - Glo assay , n = 4 from two independent experiments ( duplicate for each experiment ; mean ± SEM ) . ; HF ( 0 . 05 ) , HF 0 . 05 mg / kg ; HF ( 0 . 1 ) , HF 0 . 1 mg / kg ; DWN12088 ( 10 ) , DWN12088 10 mg / kg ; In vivo efficacy of HF and DWN12088 was determined in a bleomycin - induced lung fibrosis model . The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin ( D ) . The saturation of percutaneous oxygen ( SpO2 ) ( E ) was determined as a measurement of lung function . BLM , bleomycin ; ; HF ( 0 . 05 ) , HF 0 . 05 mg / kg ; HF ( 0 . 1 ) , HF 0 . 1 mg / kg ; DWN12088 ( 10 ) , DWN12088 10 mg / kgIn vivo efficacy of HF and DWN12088 was determined in a bleomycin - induced lung fibrosis model . The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin The collagen level was determined by hydroxyproline assay ( G ) BLM , bleomycin HF ( 0 . 05 ) , HF 0 . 05 mg / kg ; HF ( 0 . 1 ) , HF 0 . 1 mg / kg ; DWN12088 ( 10 ) , DWN12088 10 mg / kg ; In vivo efficacy of HF and DWN12088 was determined in a bleomycin - induced lung fibrosis model . The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin ( D ) . Masson ' s trichrome staining of lung tissues . ; HF ( 0 . 05 ) , HF 0 . 05 mg / kg ; HF ( 0 . 1 ) , HF 0 . 1 mg / kg ; DWN12088 ( 10 ) , DWN12088 10 mg / kg ; b , bronchiole ; v , blood vessel .

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 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 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

Figure 1Dose-response curve of HF and DWN12088 in WI-26 VA4 cells. The IC50 value for collagen level was measured as a representative of chemical efficacy. The cells were starved of serum for 6 hr and then incubated with the indicated concentrations of compounds in the presence of TGF-β (2 ng/ml) for 72 hr. The secreted pro-collagen I levels were determined by ELISA. The CC50 values were determined by CellTiter-Glo assay. Dose-response curves were determined by combining at least 2 biologically independent experiments. The IC50 values were calculated using GraphPad Prism 7.0 (Collagen level, n = 5 from three independent experiments (singlicate for one experiment and duplicate for two experiments; mean ± SEM); Celltiter-Glo assay, n = 4 from two independent experiments (duplicate for each experiment; mean ± SEM). ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). The saturation of percutaneous oxygen (SpO2) (E) was determined as a measurement of lung function. BLM, bleomycin; ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kgIn vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin The collagen level was determined by hydroxyproline assay (G) BLM, bleomycin HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). Masson's trichrome staining of lung tissues. ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg; b, bronchiole; v, blood vessel.


Figure 2WI - 26 VA4 cells were incubated in the presence of the indicated concentrations of HF and DWN12088 for 72 hr . The chemical concentrations were determined based on the IC50 for collagen level ( HF , 15 nM ; DWN12088 , 2 μM ) . The levels of proteins were determined by immunoblot assay . The proline content of the proteins based on Uniprot amino acid sequence is shown on the right side of the immunoblot images . WI - 26 VA4 cells were incubated with the indicated concentrations of HF and DWN12088 in the presence of 2 ng / ml TGF - β for 15 hr . The chemical concentrations were determined based on the IC50 values for collagen levels ( HF , 15 nM ; DWN12088 , 2 μM ) . The activation of the TGF - β pathway was determined by monitoring the phosphorylation of SMAD2 . Immunoblot images are representative of three biologically independent experiments . The band intensities of the immunoblot images in ( C ) were quantified using ImageJ ( n = 3 ; One - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; mean ± SEM ) .

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 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

Figure 2WI-26 VA4 cells were incubated in the presence of the indicated concentrations of HF and DWN12088 for 72 hr. The chemical concentrations were determined based on the IC50 for collagen level (HF, 15 nM; DWN12088, 2 μM). The levels of proteins were determined by immunoblot assay. The proline content of the proteins based on Uniprot amino acid sequence is shown on the right side of the immunoblot images.WI-26 VA4 cells were incubated with the indicated concentrations of HF and DWN12088 in the presence of 2 ng/ml TGF-β for 15 hr. The chemical concentrations were determined based on the IC50 values for collagen levels (HF, 15 nM; DWN12088, 2 μM). The activation of the TGF-β pathway was determined by monitoring the phosphorylation of SMAD2. Immunoblot images are representative of three biologically independent experiments.   The band intensities of the immunoblot images in (C) were quantified using ImageJ (n = 3; One-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM).


Figure 5The IC50 values of the indicated compounds were determined for collagen level ( HF and DWN12088 , n = 8 from four independent experiments ( duplicate for each experiment ) ; DWN11748 , n = 12 from six independent experiments ( duplicate for each experiment ) ; DWN11761 and DWN11251 , n = 6 from three independent experiments ( duplicate for each experiment ) ; mean ± SEM ) . The IC50 values of the indicated compounds were determined for cytotoxicity ( n = 4 from two independent experiments ( duplicate for each experiment ) ; mean ± SEM ) . The IC50 values of the indicated compounds were determined for TNT - luciferase activity ( n = 6 from two independent experiments ( triplicate for each experiment ) ; only for DWN11761 , n = 9 from three independent experiments ( triplicate for each experiment ) ; mean ± SEM ) . The IC50 values of the indicated compounds were determined for puromycin incorporation ( HF , n = 12 from four independent experiments ( triplicate for each experiment ) ; DWN11748 , DWN12088 , and DWN11251 , n = 9 from three independent experiments ( triplicate for each experiment ) ; DWN11761 , n = 8 from three independent experiments ( triplicate for two experiments and duplicate for one experiment ) ; mean ± SEM ) .

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Figure 5The IC50 values of the indicated compounds were determined for collagen level (HF and DWN12088, n = 8 from four independent experiments (duplicate for each experiment); DWN11748, n = 12 from six independent experiments (duplicate for each experiment); DWN11761 and DWN11251, n = 6 from three independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for cytotoxicity (n = 4 from two independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for TNT-luciferase activity (n = 6 from two independent experiments (triplicate for each experiment); only for DWN11761, n = 9 from three independent experiments (triplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for puromycin incorporation (HF, n = 12 from four independent experiments (triplicate for each experiment); DWN11748, DWN12088, and DWN11251, n = 9 from three independent experiments (triplicate for each experiment); DWN11761, n = 8 from three independent experiments (triplicate for two experiments and duplicate for one experiment); mean ± SEM).


Figure 6The binding of HF and DWN12088 to in vitro purified PARS1 WT and F1097A / E1123A / R1152L ( FA / EA / RL ) mutant was determined via microscale thermophoresis ( MST ) . Kd values are listed in the top left part of the graph ( technical replicates n = 3 ; mean ± SD ) . Catalytic activities of PARS1 WT and FA / EA / RL mutant were determined by prolylation assay in the presence of [ 3H ] proline . To monitor the effect of PARS1 RA / EA / RL mutant on the activity of PARS1 WT , the same concentrations of the proteins were pre - incubated for 10 min and then used for the assay . The formation of Pro - tRNAPro was measured using liquid scintillation counter ( technical replicates n = 3 ; mean ± SEM ) . 293T cells were transfected with the indicated plasmid DNAs for 24 hr . The cell lysates were prepared and pulled down with Strep bead for 2 hr . Co - precipitated proteins were determined by immunoblot assay . WT , PARS1 wild type ; MT , PARS1 FA / EA / RL . G , H . WI - 26 VA4 cells stably expressing each of EV ( empty vector ) or PARS1 FA / EA / RL mutant were incubated with the indicated compounds for 72 hr . Dose - response curves of the compounds for collagen levels and cytotoxicity were determined and relative ratio of CC50 to the IC50 of collagen for HF ( G ) and DWN12088 ( H ) were calculated . The IC50 values were calculated using GraphPad Prism 7 . 0 ( collagen ELISA , n = 12 from four independent experiments ( triplicate for each experiment ) ; cytotoxicity assay , n = 8 from four independent experiments ( duplicate for each experiment ) ; Welch ' s t test ; * * * P < 0 . 001 ; mean ± SEM ) . K . WI - 26 VA4 cells stably expressing each of EV or PARS1 FA / EA / RL mutant were incubated with the indicated compounds in the presence of TGF - β for 72 hr . The levels of the indicated proteins were determined by immunoblotting . The percentage of proline in polypeptide sequences is shown on the right side of immunoblot images .

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

Figure 6The binding of HF and DWN12088 to in vitro purified PARS1 WT and F1097A/E1123A/R1152L (FA/EA/RL) mutant was determined via microscale thermophoresis (MST). Kd values are listed in the top left part of the graph (technical replicates n = 3; mean ± SD).Catalytic activities of PARS1 WT and FA/EA/RL mutant were determined by prolylation assay in the presence of [3H]proline. To monitor the effect of PARS1 RA/EA/RL mutant on the activity of PARS1 WT, the same concentrations of the proteins were pre-incubated for 10 min and then used for the assay. The formation of Pro-tRNAPro was measured using liquid scintillation counter (technical replicates n = 3; mean ± SEM).293T cells were transfected with the indicated plasmid DNAs for 24 hr. The cell lysates were prepared and pulled down with Strep bead for 2 hr. Co-precipitated proteins were determined by immunoblot assay. WT, PARS1 wild type; MT, PARS1 FA/EA/RL.G, H. WI-26 VA4 cells stably expressing each of EV (empty vector) or PARS1 FA/EA/RL mutant were incubated with the indicated compounds for 72 hr. Dose-response curves of the compounds for collagen levels and cytotoxicity were determined and relative ratio of CC50 to the IC50 of collagen for HF (G) and DWN12088 (H) were calculated. The IC50 values were calculated using GraphPad Prism 7.0 (collagen ELISA, n = 12 from four independent experiments (triplicate for each experiment); cytotoxicity assay, n = 8 from four independent experiments (duplicate for each experiment); Welch's t test; ***P < 0.001; mean ± SEM).K. WI-26 VA4 cells stably expressing each of EV or PARS1 FA/EA/RL mutant were incubated with the indicated compounds in the presence of TGF-β for 72 hr. The levels of the indicated proteins were determined by immunoblotting. The percentage of proline in polypeptide sequences is shown on the right side of immunoblot images.


Figure 1A , B . HeLa cells treated with non - targeting siRNA ( Ctrl ) or C9orf72 siRNA and transfected with mCherry - EGFP - LC3 were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , bafilomycin A1 ( 100 nM ; 6 h - BafA1 ) or combinations thereof as indicated . Autophagosomes ( green + red ) and autolysosomes ( red only ) were quantified per cell ( Mean ± SEM from 3 independent experiments ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N ( cells ) = Ctrl / Ctrl : 120 ; Ctrl / Torin1 : 101 ; C9orf72 / Ctrl : 99 ; C9orf72 / Torin1 : 106 ; Ctrl / BafA1 : 116 ; Ctrl / Torin1 / BafA1 : 118 ; C9orf72 / BafA1 : 109 ; C9orf72 / Torin1 / BafA1 : 106 ) . Scale bar = 20 µm . C9orf72 knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . C , D . HEK293 cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were incubated with BafA1 , BafA1 + Torin1 ( C ) or BafA1 + rapamycin ( D ) and levels of LC3 - I and II were determined on immunoblots . Levels of LC3 - II were normalized against α - tubulin and are shown relative to the BafA1 - treated sample ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N = 3 experiments ) . E , F . Primary cortical neurons ( DIV5 / 6 ) were transfected with ECFP non - targeting ( Ctrl ) or C9orf72 miRNA ( cyan ) and EGFP - LC3 ( green ) . 3 days post transfection neurons were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , BafA1 ( 100 nM ; 5 h ) or combinations thereof as indicated . Autophagosomes were quantified as the number of EGFP - LC3 positive puncta per soma from 2 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; N ( cells ) = Ctrl miRNA / Ctrl : 77 ; Ctrl miRNA / Torin1 : 68 ; C9orf72 miRNA / Ctrl : 57 ; C9orf72 miRNA / Torin1 : 69 ; Ctrl miRNA / BafA1 : 35 ; Ctrl miRNA / Torin1 / BafA1 : 57 ; C9orf72 miRNA / BafA1 : 66 ; C9orf72 miRNA / Torin1 / BafA1 : 64 ) . Scale bar = 5 µm .

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 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 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

Figure 1A, B. HeLa cells treated with non-targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry-EGFP-LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (100 nM; 6 h - BafA1) or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (Mean ± SEM from 3 independent experiments; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 µm. C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2).C, D. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1+Torin1 (C) or BafA1+rapamycin (D) and levels of LC3-I and II were determined on immunoblots. Levels of LC3-II were normalized against α-tubulin and are shown relative to the BafA1-treated sample (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N = 3 experiments).E, F. Primary cortical neurons (DIV5/6) were transfected with ECFP non-targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP-LC3 (green). 3 days post transfection neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h) or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 µm.


Figure 2A . HEK293 cells treated with non - targeting ( Ctrl ) or FIP200 siRNA were co - transfected with EGFP - LC3 and either empty vector control ( EV ) , Myc - C9orf72S or Myc - C9orf72L . 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h . Samples were lysed and subjected to SDS - PAGE and immunoblot . Autophagy levels were determined by immunoblot for EGFP - LC3 - I and II . Expression of Myc - C9orf72 was confirmed using anti - Myc ( * indicates a nonspecific band ) . FIP200 knockdown was confirmed using anti - FIP200 antibodies . α - tubulin was used as loading control . B . HeLa cells treated with non - targeting ( Ctrl ) or FIP200 siRNA were co - transfected with empty vector ( EV ) , Myc - C9orf72S or Myc - C9orf72L ( red ) and EGFP - LC3 ( green ) to label autophagosomes . As positive control EV transfected cells were treated for 3 h with Torin1 ( 250 nM ) . Autophagy was quantified as the number of EGFP - LC3 positive autophagosomes per cell from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : * * * * P ≤ 0 . 0001 , N ( cells ) = Ctrl / EV : 73 ; FIP200 / EV : 76 ; Ctrl / EV / Torin1 : 73 , FIP200 / EV / Torin1 : 70 ; Ctrl / C9orf72L : 71 ; FIP200 / C9orf72L : 74 ; Ctrl / C9orf72S : 72 ; FIP200 / C9orf72S : 72 ) . Scale bar = 20 µm . FIP200 knockdown was confirmed by immunoblot ( Appendix Fig S2 ) .

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 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

Figure 2A. HEK293 cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with EGFP-LC3 and either empty vector control (EV), Myc-C9orf72S or Myc-C9orf72L. 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS-PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP-LC3-I and II. Expression of Myc-C9orf72 was confirmed using anti-Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti-FIP200 antibodies. α-tubulin was used as loading control.B. HeLa cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with empty vector (EV), Myc-C9orf72S or Myc-C9orf72L (red) and EGFP-LC3 (green) to label autophagosomes. As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP-LC3 positive autophagosomes per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: **** P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 µm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2).


Figure 3A , B , C . Cell lysates of HEK293 cells co - transfected with FLAG - FIP200 ( A ) and either empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc ( A and B ) antibodies . Immune pellets were probed for Myc - C9orf72 ( A and B ) , FLAG - FIP200 ( A ) on immunoblots . A , B , C . Cell lysates of HEK293 cells co - transfected with HA - ULK1 ( B ) and either empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc ( A and B ) antibodies . Immune pellets were probed for Myc - C9orf72 ( A and B ) , HA - ULK1 ( B ) on immunoblots . A , B , C . Cell lysates of HEK293 cells co - transfected with Myc - ATG13 ( C ) and either empty vector control , FLAG - C9orf72S or FLAG - C9orf72L were subjected to immunoprecipitation with anti - FLAG ( C ) antibodies . Immune pellets were probed for FLAG - C9orf72 ( C ) , Myc - ATG13 ( C ) on immunoblots . D , E . Cell lysates of HEK293 cells transfected with empty vector control , Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc antibodies Immune pellets were probed for Myc - C9orf72 and endogenous FIP200 , ULK1 , and ATG13 . There are multiple alternatively spliced forms of ATG13 ( Jung et al . , 2009 ; Alers et al . , 2011 ) ; ATG13 * is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72 . F , G , H . HeLa cells transfected with Flag - FIP200 and Myc - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( F ) C9orf72S : 22 ; C9orf72L : 18 ; EV + FIP200 : 18 ; C9orf72S + FIP200 : 18 ; C9orf72L + FIP200 : 17 . Scale bar = 10 µm ; see also Fig EV2 . F , G , H . HeLa cells transfected with HA - ULK1 and Myc - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( G ) C9orf72S : 21 ; C9orf72L : 20 ; EV + ULK1 : 20 ; C9orf72S + ULK1 : 22 ; C9orf72L + ULK1 : 20 . Scale bar = 10 µm ; see also Fig EV2 . F , G , H . HeLa cells transfected with Myc - ATG13 and EGFP - C9orf72 were fixed and processed for PLA analysis . Transfections were laced with mVenus to enable identification of transfected cells for analysis where required ( green ) . PLA signals ( red ) were counted per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ; N ( cells ) = ( H ) C9orf72S : 11 ; C9orf72L : 10 ; EV + ATG13 : 11 ; C9orf72S + ATG13 : 11 ; C9orf72L + ATG13 : 11 ) . Scale bar = 10 µm ; see also Fig EV2 . I , J , K . 35S - radiolabeled recombinant FIP200 - 6xHis segments ( I ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) . I , J , K . 35S - radiolabeled recombinant Myc - ATG13 ( J ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) . I , J , K . 35S - radiolabeled recombinant HA - ULK1 ( K ) were added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant proteins were visualized by phosphoimager ( top panels ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panels ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S2 ) .

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

Figure 3A, B, C. Cell lysates of HEK293 cells co-transfected with FLAG-FIP200 (A) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), FLAG-FIP200 (A) on immunoblots.A, B, C. Cell lysates of HEK293 cells co-transfected with HA-ULK1 (B) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), HA-ULK1 (B) on immunoblots.A, B, C. Cell lysates of HEK293 cells co-transfected with Myc-ATG13 (C) and either empty vector control, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG (C) antibodies. Immune pellets were probed for FLAG-C9orf72 (C), Myc-ATG13 (C) on immunoblots.D, E. Cell lysates of HEK293 cells transfected with empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibodies Immune pellets were probed for Myc-C9orf72 and endogenous FIP200, ULK1, and ATG13. There are multiple alternatively spliced forms of ATG13 (Jung et al., 2009; Alers et al., 2011); ATG13* is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72.F, G, H. HeLa cells transfected with Flag-FIP200 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (F) C9orf72S: 22; C9orf72L: 18; EV+FIP200: 18; C9orf72S+FIP200: 18; C9orf72L+FIP200: 17. Scale bar = 10 µm; see also Fig EV2.F, G, H. HeLa cells transfected with HA-ULK1 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (G) C9orf72S: 21; C9orf72L: 20; EV+ULK1: 20; C9orf72S+ULK1: 22; C9orf72L+ULK1: 20. Scale bar = 10 µm; see also Fig EV2.F, G, H. HeLa cells transfected with Myc-ATG13 and EGFP-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (H) C9orf72S: 11; C9orf72L: 10; EV+ ATG13: 11; C9orf72S+ATG13: 11; C9orf72L+ATG13: 11). Scale bar = 10 µm; see also Fig EV2.I, J, K. 35S-radiolabeled recombinant FIP200-6xHis segments (I) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).I, J, K. 35S-radiolabeled recombinant Myc-ATG13 (J) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).I, J, K. 35S-radiolabeled recombinant HA-ULK1 (K) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).


Figure 4A . HEK293 cells were transfected with non - targeting ( Ctrl ) or C9orf72 siRNA . Cells were treated with rapamycin for 6 h to induce autophagy . Activation of ULK1 was determined on immunoblots using phospho - ULK1 ( Ser757 ) , total ULK1 and GAPDH Abs ( loading control ) . B . HeLa cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were transfected with mCherry - FIP200 . 24 h post transfection cells were treated for 3 h with Torin1 ( 250 nM ) or vehicle ( Ctrl ) . Translocation of the ULK1 complex was quantified as the number of mCherry - FIP200 positive puncta per cell from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , ns : not significant , * * * * P ≤ 0 . 0001 ; N ( cells ) = Ctrl / Ctrl : 65 ; Ctrl / Torin1 : 60 ; C9orf72 / Ctrl : 54 ; C9orf72 / Torin1 : 49 ) . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm . C . Primary cortical neurons ( DIV5 / 6 ) were transfected with EmGFP non - targeting ( Ctrl ) or C9orf72 miRNA ( green ) and mCherry - FIP200 ( red ) ; for rescue experiments the cells were additionally transfected with mCerulean - tagged C9orf72s and C9orf72L ( cyan ) . 3 days post transfection neurons were treated for 3 h with Torin1 ( 250 nM ) or vehicle ( Ctrl ) . Translocation of the ULK1 complex was quantified as the number of mCherry - FIP200 positive puncta per soma from 2 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , ns : not significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; N ( cells ) = Ctrl miRNA / Ctrl : 134 ; Ctrl miRNA / Torin1 : 125 ; C9orf72 miRNA / Ctrl : 101 ; C9orf72 miRNA / Torin1 : 78 ; C9orf72 miRNA + C9orf72L + C9orf72S : 41 ; C9orf72 miRNA + C9orf72L + C9orf72S / Torin1 : 39 ) . Scale bar = 5 µm . D . HeLa cells were co - transfected with mCherry - FIP200 ( red ) and empty vector ( EV ) , FLAG - C9orf72L or FLAG - C9orf72S ( green ) . As positive control EV transfected cells were treated for 3 h with Torin1 ( 250 nM ) . Translocation of the ULK1 complex was quantified as the number of mCherry - FIP200 positive puncta per cell from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ) ; N ( cells ) = EV : 47 , EV + Torin1 : 31 , C9orf72L : 46 , C9orf72S : 45 ) . Scale bar = 10 µm .

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

Figure 4A. HEK293 cells were transfected with non-targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho-ULK1 (Ser757), total ULK1 and GAPDH Abs (loading control).B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry-FIP200. 24 h post transfection cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, **** P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.C. Primary cortical neurons (DIV5/6) were transfected with EmGFP non-targeting (Ctrl) or C9orf72 miRNA (green) and mCherry-FIP200 (red); for rescue experiments the cells were additionally transfected with mCerulean-tagged C9orf72s and C9orf72L (cyan). 3 days post transfection neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 µm.D. HeLa cells were co-transfected with mCherry-FIP200 (red) and empty vector (EV), FLAG-C9orf72L or FLAG-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, *** P ≤ 0.001, **** P ≤ 0.0001); N (cells) = EV: 47, EV + Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 µm.