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 .

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

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 ) .

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

G, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O

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.


A , 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 ) .

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

A, 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 ) .

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

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

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.


A . 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 .

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

A. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


A , 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 .

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

A, B, 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 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 .

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

A, B, 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 .

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

D, E. 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 .

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

F, 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 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 .

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

F, 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 ) .

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

I, 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 ) .

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

I, 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 ) .

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).


A . 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 ) .

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

A. 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 .

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

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 .

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

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

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.


A , B . HeLa cells ( A ) or SH - SY5Y neuroblastoma cells ( B ) treated with non - targeting ( Ctrl ) or Rab1a siRNA were co - transfected with mCherry - FIP200 ( red ) and empty vector ( EV ) , Myc - C9orf72L or Myc - 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 ( A , HeLa , Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test ; ns , not significant ; * * * * P ≤ 0 . 0001 ; N ( cells from 3 independent experiments ) = Ctrl / EV : 81 ; Ctrl / EV / Torin1 : 48 ; Ctrl / C9orf72L : 86 ; Ctrl / C9orf72S : 48 ; Rab1a / EV : 79 ; Rab1a / EV / Torin1 : 68 ; Rab1a / C9orf72L : 78 ; Rab1a / C9orf72S : 52 ; B , SH - SY5Y : Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test ; ns , not significant ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; N ( cells from 2 independent experiments ) = Ctrl / EV : 70 ; Ctrl / EV / Torin1 : 56 ; Ctrl / C9orf72L : 45 ; Ctrl / C9orf72S : 43 ; Rab1a / EV : 63 ; Rab1a / EV / Torin1 : 55 ; Rab1a / C9orf72L : 44 ; Rab1a / C9orf72S : 37 ) . Rab1a knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

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

A, B. HeLa cells (A) or SH-SY5Y neuroblastoma cells (B) treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV), Myc-C9orf72L or Myc-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 (A, HeLa, Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; **** P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH-SY5Y: Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; ** P ≤ 0.01; **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


C . SH - SY5Y neuroblastoma cells treated with non - targeting ( Ctrl ) or Rab1a siRNA were co - transfected with EGFP - LC3 ( green ) and empty vector ( EV ) , Myc - C9orf72L or Myc - C9orf72S ( red ) . As positive control EV transfected cells were treated for 3 h with Torin1 ( 250 nM ) . Autophagosomes were quantified as the number of EGFP - LC3 positive puncta per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test ; ns , not significant ; * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; N ( cells from 2 independent experiments ) = Ctrl / EV : 102 ; Ctrl / EV / Torin1 : 92 ; Ctrl / C9orf72L : 97 ; Ctrl / C9orf72S : 76 ; Rab1a / EV : 102 ; Rab1a / EV / Torin1 : 107 ; Rab1a / C9orf72L : 101 ; Rab1a / C9orf72S : 87 ) . Rab1a knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

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

C. SH-SY5Y neuroblastoma cells treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (red). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 102; Ctrl/EV/Torin1: 92; Ctrl/C9orf72L: 97; Ctrl/C9orf72S: 76; Rab1a/EV: 102; Rab1a/EV/Torin1: 107; Rab1a/C9orf72L: 101; Rab1a/C9orf72S: 87). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


B . Cell lysates of HEK293 cells co - transfected with Myc - Rab1a and either empty vector , FLAG - C9orf72S or FLAG - C9orf72L were subjected to immunoprecipitation with anti - FLAG antibody . Bound protein was eluted from beads using excess FLAG peptide . Immune eluates were probed for FLAG - C9orf72 and Myc - Rab1a on immunoblots . The input levels of FLAG - C9orf72 and Myc - Rab1a in the transfected cells are shown ( Inputs ) . * indicates remaining Myc - Rab1a signal after reprobing for FLAG - C9orf72 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cell lysates of HEK293 cells co-transfected with Myc-Rab1a and either empty vector, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG antibody. Bound protein was eluted from beads using excess FLAG peptide. Immune eluates were probed for FLAG-C9orf72 and Myc-Rab1a on immunoblots. The input levels of FLAG-C9orf72 and Myc-Rab1a in the transfected cells are shown (Inputs).* indicates remaining Myc-Rab1a signal after reprobing for FLAG-C9orf72.


C . Cell lysates of HEK293 cells transfected with Myc - C9orf72S or Myc - C9orf72L were subjected to immunoprecipitation with anti - Myc antibody . The resulting immune pellet was probed for endogenous Rab1a .

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

C. Cell lysates of HEK293 cells transfected with Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibody. The resulting immune pellet was probed for endogenous Rab1a.


D . 35S - radiolabelled recombinant Myc - Rab1a protein loaded with vehicle , GDP or GMP - PNP was added to GST , GST - C9orf72S and GST - C9orf72L immobilized on glutathione - coated beads . 35S - radiolabelled recombinant Myc - Rab1a protein was visualized by autoradiography ( top panel ) . Coomassie - stained GST , GST - C9orf72S and GST - C9orf72L in the pull - down samples are shown ( bottom panel ) . The identity of the Coomassie protein bands was confirmed by mass spectrometry ( # = E . coli DnaK Chaperonin ; * = E . coli 60kD Chaperonin ; Appendix Fig S3 ) . Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test ; ns , not significant ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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

D. 35S-radiolabelled recombinant Myc-Rab1a protein loaded with vehicle, GDP or GMP-PNP was added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant Myc-Rab1a protein was visualized by autoradiography (top panel). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panel). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S3). Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


E . Increasing volumes of 35S - radiolabelled recombinant Myc - C9orf72L protein were incubated with equal amounts of GST - Rab1a immobilized on glutathione - coated beads in an equilibrium binding experiment . 8 µl of 35S - radiolabelled recombinant Myc - C9orf72L protein was incubated with GST as a negative control . Bound 35S - radiolabelled Myc - C9orf72L protein was visualized by phosphoimager . Coomassie stained GST - Rab1a and GST in the pull down samples are shown . Densitometry analysis of the amount of 35S - radiolabelled recombinant Myc - C9orf72L protein bound to GST - Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola ( R2 = 0 . 94 ) .

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

E. Increasing volumes of 35S-radiolabelled recombinant Myc-C9orf72L protein were incubated with equal amounts of GST-Rab1a immobilized on glutathione-coated beads in an equilibrium binding experiment. 8 µl of 35S-radiolabelled recombinant Myc-C9orf72L protein was incubated with GST as a negative control. Bound 35S-radiolabelled Myc-C9orf72L protein was visualized by phosphoimager. Coomassie stained GST-Rab1a and GST in the pull down samples are shown. Densitometry analysis of the amount of 35S-radiolabelled recombinant Myc-C9orf72L protein bound to GST-Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola (R2=0.94).


A . HEK293 cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were transfected with HA - ULK1 , Myc - Rab1a or HA - ULK1 + Myc - Rab1a as indicated . Transfections were laced with mVenus to enable identification of transfected cells for analysis ( green ) . Transfected cells were probed with anti - HA and anti - Myc antibodies and processed for PLA . PLA proximity signals ( red ) per cell were determined from 3 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * * * P ≤ 0 . 0001 ) ; N ( cells ) = Ctrl / HA - ULK1 : 125 , Ctrl / Myc - Rab1a : 149 , Ctrl / HA - ULK1 + Myc - Rab1a : 163 , C9orf72 / HA - ULK1 : 136 , C9orf72 / Myc - Rab1a : 133 , C9orf72 / HA - ULK1 + Myc - Rab1a : 155 ) . Scale bar = 20µm .

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

A. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with HA-ULK1, Myc-Rab1a or HA-ULK1+Myc-Rab1a as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with anti-HA and anti-Myc antibodies and processed for PLA. PLA proximity signals (red) per cell were determined from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001); N (cells) = Ctrl/HA-ULK1: 125, Ctrl/Myc-Rab1a: 149, Ctrl/HA-ULK1+Myc-Rab1a: 163, C9orf72/HA-ULK1: 136, C9orf72/Myc-Rab1a: 133, C9orf72/HA-ULK1+Myc-Rab1a: 155). Scale bar = 20µm.


B . HeLa cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were co - transfected with mCherry - FIP200 ( red ) and empty vector ( EV ) or Myc - Rab1a ( Q70L ) ( green ) . Translocation of the ULK1 complex was quantified as the number of mCherry - FIP200 positive puncta per cell from 4 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 ) ; N ( cells ) = Ctrl / EV : 101 ; Ctrl / Q70L : 106 ; C9orf72 / Q70L : 42 . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

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

B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV) or Myc-Rab1a(Q70L) (green). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 4 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, * P ≤ 0.05, **** P ≤ 0.0001); N (cells) = Ctrl/EV: 101; Ctrl/Q70L: 106; C9orf72/Q70L: 42. C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


C . HeLa cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were co - transfected with EGFP - LC3 ( green ) and empty vector ( EV ) or Myc - Rab1a ( Q70L ) ( red ) . Autophagosomes were quantified as the number of EGFP - LC3 positive puncta per cell ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 ) ; N ( cells ) = Ctrl / EV : 44 ; Ctrl / Q70L : 32 ; C9orf72 / Q70L : 47 . C9orf72 knockdown was determined by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

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

C. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV) or Myc-Rab1a(Q70L) (red). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01, **** P ≤ 0.0001); N (cells) = Ctrl/EV: 44; Ctrl/Q70L: 32; C9orf72/Q70L: 47. C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


A . HeLa cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were immunostained for endogenous p62 . Accumulation of p62 was quantified by counting p62 positive puncta per cell from 3 independent experiments ( Mean ± SEM ; unpaired t - test , * * * * P ≤ 0 . 0001 ) ; N ( cells ) = Ctrl : 74 , C9orf72 : 55 ) . C9orf72 knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . Scale bar = 10 µm .

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

A. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were immunostained for endogenous p62. Accumulation of p62 was quantified by counting p62 positive puncta per cell from 3 independent experiments (Mean ± SEM; unpaired t-test, **** P ≤ 0.0001); N (cells) = Ctrl: 74, C9orf72: 55). C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.


B . Primary cortical neurons ( DIV5 ) were transduced with 4TU / cell EmGFP non - targeting control miRNA ( Ctrl ) or C9orf72 miRNA ( green ) ; for rescue experiments the cells were additionally transduced with 4TU / cell mVenus - tagged C9orf72s and C9orf72L ( verified by immunoblot , Appendix Fig S2 ) . Neurons were immunostained for endogenous p62 3 days post transduction . Accumulation of p62 was quantified by counting p62 positive puncta per soma from 2 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test , * * P ≤ 0 . 01 ; N ( cells ) = Ctrl miRNA : 80 ; C9orf72 miRNA : 80 ; C9orf72 miRNA + C9orf72L + C9orf72S : 75 ) . Scale bar = 10 µm .

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

B. Primary cortical neurons (DIV5) were transduced with 4TU/cell EmGFP non-targeting control miRNA (Ctrl) or C9orf72 miRNA (green); for rescue experiments the cells were additionally transduced with 4TU/cell mVenus-tagged C9orf72s and C9orf72L (verified by immunoblot, Appendix Fig S2). Neurons were immunostained for endogenous p62 3 days post transduction. Accumulation of p62 was quantified by counting p62 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01; N (cells) = Ctrl miRNA: 80; C9orf72 miRNA: 80; C9orf72 miRNA+C9orf72L+C9orf72S: 75). Scale bar = 10 µm.


A , B . Two C9ALS / FTD iNeuron cultures ( 183 and 201 ) and their matching controls ( 170 and 155 ) were treated with vehicle ( Ctrl ) or bafilomycin A1 ( 100 nM ; 6 h - BafA1 ) and processed for immunoblot detection of LC3 . LC3 - II levels were normalized to α - Tubulin ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test ; ns , not significant ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; Ctrl 209 / Pat 201 , n = 4 ; Ctrl 155 / Pat 183 , n = 6 ) .

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

A, B. Two C9ALS/FTD iNeuron cultures (183 and 201) and their matching controls (170 and 155) were treated with vehicle (Ctrl) or bafilomycin A1 (100 nM; 6 h - BafA1) and processed for immunoblot detection of LC3. LC3-II levels were normalized to α-Tubulin (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; Ctrl 209/Pat 201, n=4; Ctrl 155/Pat 183, n=6).


( A ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies . Scale bar : 200 nm .

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

(A) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies. Scale bar: 200 nm.


( B ) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / ODF2 fluorescence ratio in the indicated cell lines and genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed for each condition , a . u . = arbitrary units . Unpaired Mann - Whitney test ( * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/ODF2 fluorescence ratio in the indicated cell lines and genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Mann-Whitney test (****P < 0.0001).


( C ) Representative fluorescence micrographs from the indicated cell lines co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Co - localization between ODF2 and PIDD1 ( n = 11 ) , FBF1 and PIDD1 ( n = 21 ) , and ANKRD26 and PIDD1 ( n = 17 ) assessed on images The value represents the fraction PIDD1 objects touching either ODF2 , FBF1 or ANKRD26 objects .

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

(D) Co-localization between ODF2 and PIDD1 (n = 11), FBF1 and PIDD1 (n = 21), and ANKRD26 and PIDD1 (n = 17) assessed on images The value represents the fraction PIDD1 objects touching either ODF2, FBF1 or ANKRD26 objects.


( E ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies , scale bar : 200 nm .

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

(E) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies, scale bar: 200 nm.


( F ) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( G ) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / CEP128 fluorescence ratio in RPE1 of the indicated genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed from as many individual cells for each condition , a . u . = arbitrary units . Statistical significance was assessed by Kruskal - Wallis test , comparing each sample to the wild type ( * * * * P < 0 . 0001 ) .

(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).


( B ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(B) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( C ) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

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

(C) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( D ) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

(D) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( E ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h . A fraction of DHCB treated cells were released into fresh medium for other 24h ( release ) . Samples were subjected to immunoblotting ; n = 3 independent experiments .

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

(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.


( B ) Fluorescence micrographs of RPE1 cells of the indicated genotypes , either treated for 24h with DHCB or with vehicle alone ( DMSO ) . Blow - up without Hoechst 33342 is magnified 2 . 5X . Scale bar : 5 μm . ( C ) Immunofluorescence micrographs of RPE1 as in ( B ) were used to visually assess the percentage of cells presenting one or two nuclei . N = 3 , ≥ 50 cells from each independent experiment . Mean values ± s . e . m . are reported . The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed ( ANOVA test ; * P < 0 . 05 ) . ( D ) Immunofluorescence micrographs of RPE1 as in ( B ) were used to visual score the number of centrosomes per cell by counting the number of γ - tubulin - positive centrioles . N = 3 , ≥ 50 cells from each independent experiment . Mean values ± s . e . m . are reported . The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed ( ANOVA test ; * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence micrographs of RPE1 cells of the indicated genotypes, either treated for 24h with DHCB or with vehicle alone (DMSO). Blow-up without Hoechst 33342 is magnified 2.5X. Scale bar: 5 μm. (C) Immunofluorescence micrographs of RPE1 as in (B) were used to visually assess the percentage of cells presenting one or two nuclei. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed (ANOVA test; *P < 0.05). (D) Immunofluorescence micrographs of RPE1 as in (B) were used to visual score the number of centrosomes per cell by counting the number of γ-tubulin-positive centrioles. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed (ANOVA test; **P < 0.01).


( E ) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment , inferred on the basis of the increase of ploidies ≥ 4C ( upper panel ) , and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment ( lower panel ) . Individual values of biological replicates , their mean and standard deviations are reported .

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

(E) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment, inferred on the basis of the increase of ploidies ≥ 4C (upper panel), and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment (lower panel). Individual values of biological replicates, their mean and standard deviations are reported.


( F ) RPE1 cells were either left untransduced ( mock ) or transduced with lentiviral vectors expressing the indicated Myc - ANKRD26 constructs . Subsequently , cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting ; n = 3 independent experiments .

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

(F) RPE1 cells were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated Myc-ANKRD26 constructs. Subsequently, cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting; n = 3 independent experiments.


( B ) A549 cells of the indicated genotypes were either left untransduced ( mock ) or transduced with lentiviral vectors expressing the indicated PIDD1 - V5 derivatives and subjected to immunoblotting . N = 2 independent experiments .

O O O O O O O O O O O O O O O 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) A549 cells of the indicated genotypes were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated PIDD1-V5 derivatives and subjected to immunoblotting. N = 2 independent experiments.


( C ) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced ( mock ) , or transduced with PIDD1 - V5 lentiviral vectors and co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . ( E ) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units .

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

(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units.


( F ) Representative fluorescence micrographs of A549 cells of the indicated genotypes . Cells were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors expressing the PIDD1 non - cleavable derivative ( PIDD1S446A - S588A ) or truncations thereof . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1 - V5 in its wild type form . Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting . N = 2 independent experiments .

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

(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( B , C ) A549 cells of the indicated genotypes were either left untransduced ( mock ) , or transduced with lentiviral vectors expressing PIDD1 - V5 in its wild type form or carrying the indicated point mutations . Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting . N = 2 independent experiments .

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

(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( D ) Fluorescence micrographs of A549 cells of the indicated genotypes . Cells were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors carrying the indicated point mutations , and stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1 - GFP . Centrosomal antigens were stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


The centrioles subjected to fluorescence intensity measurement in ( B ) are labelled with numbers . ( B ) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase , a . u . = arbitrary units .

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

The centrioles subjected to fluorescence intensity measurement in (B) are labelled with numbers. (B) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase, a.u. = arbitrary units.


( C ) Fluorescence micrographs of RPE1 cells stably expressing CETN1 - GFP treated either with DMSO or with DHCB for 24h . Blow - ups 33342 are magnified 2 . 5X . Scale bar : 5 μm . ( D ) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in ( C ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition , a . u . = arbitrary units .

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

(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units.


( E ) Movie stills of a representative RPE1 cell stably expressing CETN1 - GFP treated with DHCB and subjected to time - lapse video microscopy in the presence of SiR - DNA . Time is expressed in minutes , relative to anaphase onset . The dashed line indicates the plasma membrane of the cell of interest , arrowheads indicate the centrosomes ' position .

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

(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position.


( F ) Centrosomal distance over time in RPE1 cells stably expressing CETN1 - GFP and treated with DHCB . Time zero corresponds to the frame preceding anaphase onset . Coloured dots ( lower panel ) summarize the clustering time for each cell , mean ± standard deviation in black . Data calculated from four - dimensional imaging as in ( E ) . N = 10 cells .

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

(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.


( A ) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments ( ZM = ZM447439 ; REV = reversine ) . Nocodazole concentrations are 0 . 03 μM , 0 . 1 μM , 0 . 33 μM , 1 μM , 3 . 3 μM . Median ( red ) and 95 % confidence interval thereof ( black ) are shown . N > 50 cells were analysed . ( B ) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in ( A ) . N = 3 independent experiments . ( C ) DNA content analysis of A549 cells subjected to the indicated treatments as in ( A ) either for 24h ( left panels ) or 48h ( right panels ) . N = 2 independent experiments .

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

(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments.


(E) Immunoblots of RPE1 cells synchronized


( F ) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.


( A ) A549 cells of the indicated genotypes were treated for 24h as indicated ( CPT = camptothecin , ZM = ZM447439 ) . Samples were subjected to immunoblotting ; n = 3 independent experiments . ( B ) A549 cells treated as in ( A ) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ - tubulin - positive centrioles per cell . N = 3 , ≥ 50 cells from each independent experiment .

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

(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment.


( C ) RT - qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . The average fold of induction ± standard deviation is shown . N = 3 biological replicates with two technical replicates each . Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type ( WT )

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

(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells


with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . ( D ) Immunoblot analysis of samples treated N = 3 independent experiments .

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

with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments.


a ) Rapamycin ( Rap ) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK - 293T cells . ( n = 3 ; two asterisks , P 0 . 01 ) . DMSO , dimethylsulphoxide .

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

a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells. (n = 3; two asterisks, P 0.01). DMSO, dimethylsulphoxide.


( b ) Expression analysis of Wnt target genes by quantitative RT - PCR after 4 h of treatment with rapamycin . ( n = 3 ; asterisk , P 0 . 05 ; two asterisks , P 0 . 01 ) . Wnt3a CM , Wnt3a conditioned medium .

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

(b) Expression analysis of Wnt target genes by quantitative RT-PCR after 4 h of treatment with rapamycin. (n = 3; asterisk, P 0.05; two asterisks, P 0.01). Wnt3a CM, Wnt3a conditioned medium.


( c ) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling . ( n = 3 ; asterisk , P 0 . 05 ) .

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

(c) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling. (n = 3; asterisk, P 0.05).


( d ) Atg5 knockout enhanced Wnt signalling . TopFlash reporter activity induced by Wnt3a CM in Atg5 − / − or wild - type MEFs . Results are means ± s . d . after normalization to Renilla luciferase activity . ( n = 3 ; asterisk , P 0 . 05 ) .

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

(d) Atg5 knockout enhanced Wnt signalling. TopFlash reporter activity induced by Wnt3a CM in Atg5−/− or wild-type MEFs. Results are means ± s.d. after normalization to Renilla luciferase activity. (n = 3; asterisk, P 0.05).


( a ) Dvl2 underwent autophagy - mediated degradation . E - cadherin was used as a loading control for membrane fraction , and tubulin as a loading control for cytosolic proteins . The asterisk indicates the phosphorylated form of Dvl2 .

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(a) Dvl2 underwent autophagy-mediated degradation. E-cadherin was used as a loading control for membrane fraction, and tubulin as a loading control for cytosolic proteins. The asterisk indicates the phosphorylated form of Dvl2.


( b ) The half - life of Dvl2 was prolonged in Atg5 − / − cells . MEFs were subjected to starvation ( starv . ) for the indicated durations and harvested for immunoblotting with the indicated antibodies . Dvl2 levels were quantified ( right panel ) . Autophagy induction is indicated by the levels of LC3 II and p62 degradation .

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(b) The half-life of Dvl2 was prolonged in Atg5−/− cells. MEFs were subjected to starvation (starv.) for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). Autophagy induction is indicated by the levels of LC3 II and p62 degradation.


( c ) Reintroduction of Atg5 in Atg5 − / − MEFs restored starvation - induced Dvl2 degradation . MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for the indicated durations , and the cell lysates were subjected to immunoblotting with the indicated antibodies .

(c) Reintroduction of Atg5 in Atg5−/− MEFs restored starvation-induced Dvl2 degradation. MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for the indicated durations, and the cell lysates were subjected to immunoblotting with the indicated antibodies.


( d ) Dvl2 degradation was slowed down in Atg7 − / − MEFs . MEFs were subjected to starvation for the indicated durations and harvested for immunoblotting with the indicated antibodies . Dvl2 levels were quantified ( right panel ) .

(d) Dvl2 degradation was slowed down in Atg7−/− MEFs. MEFs were subjected to starvation for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel).


( e ) The continued degradation of Dvl2 in Atg5 − / − cells is proteasome - dependent . MEFs were starved for 2 , 4 or 8 h with MG132 ( 1 μM ) or BFA1 ( 0 . 1 μM ) for different durations , followed by anti - Dvl2 immunoblotting to detect endogenous Dvl2 protein levels . Tubulin served as a loading control . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(e) The continued degradation of Dvl2 in Atg5−/− cells is proteasome-dependent. MEFs were starved for 2, 4 or 8 h with MG132 (1 μM) or BFA1 (0.1 μM) for different durations, followed by anti-Dvl2 immunoblotting to detect endogenous Dvl2 protein levels. Tubulin served as a loading control. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Immunoelectron microscopy analysis of HeLa cells transfected with Flag - Dvl2 under nutrient - rich or starvation conditions . The arrowheads indicate Flag - Dvl2 . Scale bars , 1 μm ( left ) ; 250 nm ( right ) .

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(a) Immunoelectron microscopy analysis of HeLa cells transfected with Flag-Dvl2 under nutrient-rich or starvation conditions. The arrowheads indicate Flag-Dvl2. Scale bars, 1 μm (left); 250 nm (right).


( b ) Localization of endogenous Dvl proteins ( red ) and GFP - LC3 ( green ) in GFP - LC3 stable HeLa cells in nutrient - rich medium or under starvation ( starv . ) conditions with BFA1 for 4 h . The right panel shows the percentage co - localization between Dvls and GFP - LC3 - positive dots . Scale bar , 5 μm .

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(b) Localization of endogenous Dvl proteins (red) and GFP-LC3 (green) in GFP-LC3 stable HeLa cells in nutrient-rich medium or under starvation (starv.) conditions with BFA1 for 4 h. The right panel shows the percentage co-localization between Dvls and GFP-LC3-positive dots. Scale bar, 5 μm.


( c ) Immunoelectron microscopy analysis of HeLa cells transfected with Flag - Dvl2 and GFP - LC3 under starvation conditions . Arrows indicate Flag - Dvl2 ; arrowheads indicate GFP - LC3 . Scale bar , 250 nm .

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(c) Immunoelectron microscopy analysis of HeLa cells transfected with Flag-Dvl2 and GFP-LC3 under starvation conditions. Arrows indicate Flag-Dvl2; arrowheads indicate GFP-LC3. Scale bar, 250 nm.


( d ) Dvl2 - LC3 interaction in vivo . IB , immunoblotting ; IP , immunoprecipitation ; S , starvation ; WCL , whole - cell lysate ; N , nutrient - rich .

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(d) Dvl2-LC3 interaction in vivo. IB, immunoblotting; IP, immunoprecipitation; S, starvation; WCL, whole-cell lysate; N, nutrient-rich.


( e , f ) Dvl2 interacts directly with LC3 ( e ) and GABARAP ( f ) but not with GATE - 16 in vitro . GST pulldown assay using in vitro - translated Flag - Dvl2 and immobilized GST or the indicated GST fusions . Precipitated proteins were detected with anti - Flag antibody . Coomassie staining of GST and GST - LC3 protein is shown .

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(e, f) Dvl2 interacts directly with LC3 (e) and GABARAP (f) but not with GATE-16 in vitro. GST pulldown assay using in vitro-translated Flag-Dvl2 and immobilized GST or the indicated GST fusions. Precipitated proteins were detected with anti-Flag antibody. Coomassie staining of GST and GST-LC3 protein is shown.


( g ) The LC - 3 - binding motif is important for the Dvl2 - LC3 interaction . GST - LC3 - immobilized beads were incubated with the lysates from the cells expressing wild - type ( WT ) Dvl2 or the W444A , I447A mutant ( MUT ) ; the pulldowns were then subjected to anti - haemagglutinin ( anti - HA ) immunoblotting . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(g) The LC-3-binding motif is important for the Dvl2-LC3 interaction. GST-LC3-immobilized beads were incubated with the lysates from the cells expressing wild-type (WT) Dvl2 or the W444A, I447A mutant (MUT); the pulldowns were then subjected to anti-haemagglutinin (anti-HA) immunoblotting. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Interaction between LC3 and wild - type Dvl2 or its point mutants .

(a) Interaction between LC3 and wild-type Dvl2 or its point mutants.


( b ) Ubiquitylation of wild - type Dvl2 and its variants . Transfected HEK - 293T cells were treated with BFA1 ( 0 . 2 μM ) for 6 h . Ubiquitylated proteins were precipitated with Ni - nitrilotriacetate ( Ni - NTA ) beads , followed by anti - HA immunoblotting . Ub , ubiquitin .

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(b) Ubiquitylation of wild-type Dvl2 and its variants. Transfected HEK-293T cells were treated with BFA1 (0.2 μM) for 6 h. Ubiquitylated proteins were precipitated with Ni-nitrilotriacetate (Ni-NTA) beads, followed by anti-HA immunoblotting. Ub, ubiquitin.


( c ) Degradation kinetics of wild - type Dvl2 and its variants under starvation conditions . Lower panel : quantification of the immunoblotting results .

(c) Degradation kinetics of wild-type Dvl2 and its variants under starvation conditions. Lower panel: quantification of the immunoblotting results.


( d ) Starvation promotes co - localization of Dvl , ubiquitin and p62 . Scale bar , 10 μm .

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(d) Starvation promotes co-localization of Dvl, ubiquitin and p62. Scale bar, 10 μm.


( e ) Starvation induces the formation of high - molecular - mass Dvl2 aggregates .

(e) Starvation induces the formation of high-molecular-mass Dvl2 aggregates.


( f ) p62 knockdown alters Dvl localization . Scale bar , 10 μm . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(f) p62 knockdown alters Dvl localization. Scale bar, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Knockdown of p62 expression attenuates the LC3 - Dvl2 interaction . shRNA , short hairpin RNA .

(a) Knockdown of p62 expression attenuates the LC3-Dvl2 interaction. shRNA, short hairpin RNA.


( b ) Co - localization of GFP - LC3 , p62 and Dvl in HeLa GFP - LC3 stable cells on starvation . Proteins visualized by immunofluorescence with anti - GFP ( green ) , anti - p62 ( blue ) and anti - Dvl ( red ) antibodies . Scale bar , 10 μm .

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(b) Co-localization of GFP-LC3, p62 and Dvl in HeLa GFP-LC3 stable cells on starvation. Proteins visualized by immunofluorescence with anti-GFP (green), anti-p62 (blue) and anti-Dvl (red) antibodies. Scale bar, 10 μm.


( c ) p62 knockdown slows the degradation of Dvl2 on starvation . HEK - 293T cells were transfected with p62 siRNA . At 48 h after transfection , cells were subjected to starvation for various times . The cell lysates were subjected to immunoblotting with the antibodies indicated . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(c) p62 knockdown slows the degradation of Dvl2 on starvation. HEK-293T cells were transfected with p62 siRNA. At 48 h after transfection, cells were subjected to starvation for various times. The cell lysates were subjected to immunoblotting with the antibodies indicated. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL .

(a) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL.


( b ) In vitro ubiquitylation of Dvl2 by the pVHL E3 ligase complex including pVHL , Elongin B , Elongin C , Rbx1 and Cul2 . In vitro - translated Flag - Dvl2 was incubated with or without purified E1 , E2 and E3 ligase complex , Elongin C and pVHL . The reaction was subjected to anti - Flag immunoprecipitation . Dvl2 ubiquitylation was examined by using anti - ubiquitin immunoblotting .

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(b) In vitro ubiquitylation of Dvl2 by the pVHL E3 ligase complex including pVHL, Elongin B, Elongin C, Rbx1 and Cul2. In vitro-translated Flag-Dvl2 was incubated with or without purified E1, E2 and E3 ligase complex, Elongin C and pVHL. The reaction was subjected to anti-Flag immunoprecipitation. Dvl2 ubiquitylation was examined by using anti-ubiquitin immunoblotting.


( c ) Ubiquitylation of Dvl2 is decreased by pVHL knockdown .

(c) Ubiquitylation of Dvl2 is decreased by pVHL knockdown.


( d ) Starvation elevates Dvl2 - Cul2 interaction . After co - transfection with Myc - Cul2 and Flag - Dvl2 for 36 h , HEK - 293T cells were cultured in nutrient - rich medium or starved with BFA1 ( 0 . 1 μM ) for 4 h . Cells were harvested for anti - Myc immunoprecipitation followed by anti - Flag immunoblotting . Total protein expression was confirmed by immunoblotting with whole - cell lysates .

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(d) Starvation elevates Dvl2-Cul2 interaction. After co-transfection with Myc-Cul2 and Flag-Dvl2 for 36 h, HEK-293T cells were cultured in nutrient-rich medium or starved with BFA1 (0.1 μM) for 4 h. Cells were harvested for anti-Myc immunoprecipitation followed by anti-Flag immunoblotting. Total protein expression was confirmed by immunoblotting with whole-cell lysates.


( e ) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions .

(e) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions.


( f ) Dvl2 ubiquitylation is required for its direct interaction with p62 . Dvl2 was ubiquitylated by the pVHL E3 ligase complex in vitro , followed by in vitro binding assay with recombinant p62 .

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(f) Dvl2 ubiquitylation is required for its direct interaction with p62. Dvl2 was ubiquitylated by the pVHL E3 ligase complex in vitro, followed by in vitro binding assay with recombinant p62.


( g ) pVHL knockdown decreases Dvl2 degradation under starvation conditions .

(g) pVHL knockdown decreases Dvl2 degradation under starvation conditions.


( h ) Knockdown of p62 or LC3 attenuates the pVHL - promoted Dvl2 degradation under starvation conditions . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(h) Knockdown of p62 or LC3 attenuates the pVHL-promoted Dvl2 degradation under starvation conditions. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a , b ) Depletion of Beclin 1 by shRNA promotes growth in soft agar . SW480 cells stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar and treated with or without Dkk ( a ) ; the colonies were then scored after 10 days ( b ) . Numbers in bars indicate the numbers of colonies . Results are means ± s . d . ( n = 4 ) . ( Asterisk , P 0 . 05 ) . Scale bar , 200 μm .

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(a, b) Depletion of Beclin 1 by shRNA promotes growth in soft agar. SW480 cells stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar and treated with or without Dkk (a); the colonies were then scored after 10 days (b). Numbers in bars indicate the numbers of colonies. Results are means ± s.d. (n = 4). (Asterisk, P 0.05). Scale bar, 200 μm.


( c ) Representative images from colon cancer tissues by immunohistochemistry staining . Negative correlation between Beclin1 and Dvl was observed in late stages of tumours , whereas positive correlation between p62 and Dvl in late stages of tumours . Scale bar , 25 μm .

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(c) Representative images from colon cancer tissues by immunohistochemistry staining. Negative correlation between Beclin1 and Dvl was observed in late stages of tumours, whereas positive correlation between p62 and Dvl in late stages of tumours. Scale bar, 25 μm.


( d ) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies . N , normal tissue ; T , tumour .

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(d) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies. N, normal tissue; T, tumour.


( A ) Representative flow cytometry plots ( left ) and pooled data ( right ) of competition assays with sgControl and sgRnf31 B16 - F10 ova tumor cells after culture with the indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) CRISPR / Cas9 screen showing depleted B16 - F10 genes after one week culture with TNF + IFN - γ ( both 10 ng / mL ) ; blue genes indicate overall genes with p < 0 . 001 , black genes indicate genes of interest with p < 0 . 05 . Significance assessed using MAGeCK test .

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(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.


( H ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( A ) PI staining of sgControl or sgRnf31 B16 - F10 tumor cells treated with combined TNF / IFN - γ ( both 10 ng / mL ) for 24 hours in the absence and presence of QVD - OPh ( 10 μM ) ; n . s . : no significance , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16 - F10 tumor cells cultured with the indicated cytokine ( s ) ( 10 ng / mL for all ) for 0 , 3 , 6 , or 9 hours ; β - actin was used as a loading control .

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(B) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16-F10 tumor cells cultured with the indicated cytokine(s) (10 ng/mL for all) for 0, 3, 6, or 9 hours; β-actin was used as a loading control.


( D ) CRISPR / Cas9 screen showing enriched genes from sgRnf31 B16 - F10 tumor cells after successive treatment with combined TNF / IFN - γ , as described in ( C ) ; red genes indicate overall genes with p < 0 . 0001 . Significance assessed using MAGeCK test .

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(D) CRISPR/Cas9 screen showing enriched genes from sgRnf31 B16-F10 tumor cells after successive treatment with combined TNF/IFN-γ, as described in (C); red genes indicate overall genes with p < 0.0001. Significance assessed using MAGeCK test.


( G ) PI staining of sgControl , sgRnf31 , sgRnf31 / Stat1 , sgRnf31 / Casp8 , sgRnf31 / Bid , or sgRnf31 / Rela B16 - F10 tumor cells treated with combined TNF / IFN - γ for 24 hours ; * p < 0 . 05 , * * * * p < 0 . 0001 , asterisk ( s ) indicated group vs TNF / IFN - γ - treated sgRnf31 group , Unpaired t - test for n > 3 biological replicates . Pooled data are represented as mean ± SEM .

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(G) PI staining of sgControl, sgRnf31, sgRnf31/Stat1, sgRnf31/Casp8, sgRnf31/Bid, or sgRnf31/Rela B16-F10 tumor cells treated with combined TNF/IFN-γ for 24 hours; *p < 0.05, ****p < 0.0001, asterisk(s) indicated group vs TNF/IFN-γ-treated sgRnf31 group, Unpaired t-test for n > 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of parental B16 - F10 tumor cells left untreated or treated with supernatant ( Sup ) ( derived from a previous overnight OT - I / B16 - F10 ova co - culture at the indicated E : T ratio for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( the concentration of vehicle / HOIPIN - 1 was halved upon the addition of Sup ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( C ) Representative flow cytometry plots ( left ) and pooled data ( right ) showing PI staining of parental A375 tumor cells left untreated or treated with TNF / IFN - γ ( both 10 ng / mL ) for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( the concentration of vehicle / HOIPIN - 1 was halved upon the addition of cytokines ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Unpaired t - test for n = 3 biological replicates . Pooled data are represented as mean ± SEM .

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

(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.