Figure 5A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, 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.


Figure 6B . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B. 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.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).


Figure 7A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. 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.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.


Figure 8A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. 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.


Figure 9A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A, 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).


Figure 1 ( 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 ) . ( 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 . ( 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 . ( 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 ) .

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

Figure 1(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).(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.(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.(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).


Figure 2 ( 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 ) . ( 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 .

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

Figure 2(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).(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.


Figure 3 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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). (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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.


Figure 6 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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.


Figure 7 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 8 ( 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 ) cellswith 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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) cellswith 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.


figf1a ) 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 . ( 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 ) .

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

figf1a) 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.(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).


figf2 ( 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 . ( 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 ) . ( 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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figf2(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.(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).(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.


figf3 ( 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 . ( 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 . ( 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 . ( 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 .

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

figf3(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.(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.(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.(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.


figf4 ( 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 . ( 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 . ( 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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figf4(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.(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.(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.


figf5 ( 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 . ( 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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figf5(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.(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.


figf6 ( 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 . ( 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 . ( 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 . ( 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 .

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

figf6(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.(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.(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.(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.


figf7 ( 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 . ( d ) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies . N , normal tissue ; T , tumour .

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figf7(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.(d) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies. N, normal tissue; T, tumour.


Figure 2 ( 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 . ( 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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Figure 2(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.(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.


Figure 3 ( 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 . ( 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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Figure 3(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.(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.


Figure 4 ( 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 .

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Figure 4(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.


Figure 5 ( B ) Individual ( left ) and average ( right ) in vivo growth of subcutaneous wild - type or sgRnf31 B16 - F10 ova tumors left untreated or treated with adoptively transferred OT - I CD8 + T cells ( on days indicated by black arrows ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for OT - I - treated wild - type vs OT - I - treated sgRnf31 with n = 5 / 6 mice per group . Pooled data are represented as mean ± SEM . ( C ) Overall survival of mice from ( B ) ; * p < 0 . 05 , * * p < 0 . 01 , Log - rank test .

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

Figure 5(B) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.(C) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.


Figure 1 ( E and F ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol - d6 ( 5 mM ) or ethanol - 1 - 13C ( 5 mM ) for 0 . 5 h . Relative acetate m + 3 and acetate m + 1 levels ( E - F , top panels ) were examined . Protein levels of p53 and ALDH2 ( E - F , bottom panels ) were analyzed by western blot . ( H - J ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin ( 2 ng / mL ) ( H ) nutlin3α ( 10 μM ) ( I ) or cyanamide ( 1 mM ) ( J ) for 24 h . Relative enzymatic activity of ALDH2 was examined ( top panel ) . Protein expression was determined by western blot analysis ( bottom panel ) . ( P - R ) p53 + / + and p53 - / - mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 0 . 5 h . Relative acetate m + 3 levels in mouse liver tissues ( P ) and mouse serum ( Q ) , and relative hepatic acetyl - CoA ( R ) were examined . ( S ) Mouse hepatocyte isolated from p53 + / + and p53 - / - mice and lysates were treated with or without cyanamide ( 1mM ) . Relative acetyl - CoA level ( top panel ) and protein expression ( bottom panel ) were measured . ( T ) CCC - HEL - 1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide ( 1mM ) . Relative acetyl - CoA level ( top panel ) and protein expression ( bottom panel ) were analyzed . ( U - X ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol - d6 ( 5 mM ) ( U ) or ethanol - 1 - 13C ( 5 mM ) ( X ) for 0 . 5 h . ( U ) Relative acetate m + 3 and acetyl - CoA m + 3 levels are shown . ( X ) Relative acetate m + 1 and acetyl - CoA m + 3 levels were examined . Protein levels of p53 and ALDH2 were analyzed by western blot ( V - W ) .

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

Figure 1(E and F) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol-d6 (5 mM) or ethanol-1-13C (5 mM) for 0.5 h. Relative acetate m+3 and acetate m+1 levels (E-F, top panels) were examined. Protein levels of p53 and ALDH2 (E-F, bottom panels) were analyzed by western blot.(H-J) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin (2 ng/mL) (H) nutlin3α (10 μM) (I) or cyanamide (1 mM) (J) for 24 h. Relative enzymatic activity of ALDH2 was examined (top panel). Protein expression was determined by western blot analysis (bottom panel).(P-R) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h. Relative acetate m+3 levels in mouse liver tissues (P) and mouse serum (Q), and relative hepatic acetyl-CoA (R) were examined.(S) Mouse hepatocyte isolated from p53+/+ and p53-/- mice and lysates were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were measured. (T) CCC-HEL-1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were analyzed.(U-X) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol-d6 (5 mM) (U) or ethanol-1-13C (5 mM) (X) for 0.5 h. (U) Relative acetate m+3 and acetyl-CoA m+3 levels are shown. (X) Relative acetate m+1 and acetyl-CoA m+3 levels were examined. Protein levels of p53 and ALDH2 were analyzed by western blot (V- W).


Figure 2 ( C ) Lysates from mice liver tissues ( WT , p53 - / - and ALDH2 - / - ) were immunoprecipitated using p53 or ALDH2 antibody , and bound proteins were analyzed by western blot . ( G ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 mut312 - 323 in ALDH2 - knockout cell lines . The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol ( 5 mM ) for 0 . 5 h and relative ALDH2 enzymatic activities were examined . Protein expression was analyzed by western blot . ( H ) Lysates from 293T cells transfected with HA - ALDH2 , Flag - ALDH2 and increasing amounts of GST - p53 or vector control ( - ) as indicated were immunoprecipitated using anti - Flag M2 affinity gels , and bound proteins were analyzed by western blot . ( I ) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol ( 5 mM ) for 0 . 5 h and cell lysates were treated with disuccinimidyl suberate ( DSS ) or DMSO for 0 . 5 h and protein levels of p53 and ALDH2 were examined . ( J ) Subcellular fractionation of HepG2 cells treated with doxorubicin ( 2 ng / mL ) or nutlin3α ( 10 μM ) or PFTα ( 10 μM ) for 24 h were immunoprecipitated using p53 antibody , and bound proteins were analyzed by western blot .

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

Figure 2(C) Lysates from mice liver tissues (WT, p53-/- and ALDH2-/-) were immunoprecipitated using p53 or ALDH2 antibody, and bound proteins were analyzed by western blot.(G) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 mut312-323 in ALDH2-knockout cell lines. The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol (5 mM) for 0.5 h and relative ALDH2 enzymatic activities were examined. Protein expression was analyzed by western blot.(H) Lysates from 293T cells transfected with HA-ALDH2, Flag-ALDH2 and increasing amounts of GST-p53 or vector control (-) as indicated were immunoprecipitated using anti-Flag M2 affinity gels, and bound proteins were analyzed by western blot.(I) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol (5 mM) for 0.5 h and cell lysates were treated with disuccinimidyl suberate (DSS) or DMSO for 0.5 h and protein levels of p53 and ALDH2 were examined.(J) Subcellular fractionation of HepG2 cells treated with doxorubicin (2 ng/mL) or nutlin3α (10 μM) or PFTα (10 μM) for 24 h were immunoprecipitated using p53 antibody, and bound proteins were analyzed by western blot.


Figure 3 ( A - B ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities ( A , B , top panels ) and examine p53 and ALDH2 proteins ( A , B , bottom panels ) . ( E ) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells . Cell lysates were separated by SDS - PAGE and analyzed by western blot . Actin was used as an internal control . Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures . ( F ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 - N186A , E285A , C319A in ALDH2 - knockout cell lines . Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0 . 5 hour at room temperature , followed by pronase digestion . ALDH2 proteins were analyzed by western blot . Relative ALDH2 / Actin ratios are shown . ( H - J ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 N186A in ALDH2 - knockout cell lines . The cells were treated with pyruvate ( 1 mM ) for 24 h . Relative ALDH2 activity ( H , top panel ) , acetate ( I ) and acetyl - CoA ( J ) were measured . The expression of p53 and ALDH2 were analyzed by western blot ( H , bottom panel ) .

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

Figure 3(A-B) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities (A, B, top panels) and examine p53 and ALDH2 proteins (A, B, bottom panels).(E) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells. Cell lysates were separated by SDS-PAGE and analyzed by western blot. Actin was used as an internal control. Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures.(F) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2-N186A, E285A, C319A in ALDH2-knockout cell lines. Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0.5 hour at room temperature, followed by pronase digestion. ALDH2 proteins were analyzed by western blot. Relative ALDH2/Actin ratios are shown.(H-J) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 N186A in ALDH2-knockout cell lines. The cells were treated with pyruvate (1 mM) for 24 h. Relative ALDH2 activity (H, top panel), acetate (I) and acetyl-CoA (J) were measured. The expression of p53 and ALDH2 were analyzed by western blot (H, bottom panel).


Figure 4H & E ( hematoxylin - eosin ) staining ( C , top ) and oil red O staining ( C , bottom ) were examined . Relative mRNA levels of IL - 6 , were analyzed . were examined . Data in are from n = 5 mice per group . Relative mRNA levels of , IL - 1β , TNF - α , Cxcl1 , Cxcl2 , Col1a1 were analyzed . Data in are from n = 5 mice per group . ( J ) Relative triglyceride level was examined . ( K - L ) Relative levels of serum ALT ( K ) and AST ( L ) were examined . Data in are from n = 5 mice per group . ( M - O ) WT , p53 − / − , ALDH2 − / − and p53 − / − ALDH2 − / − C57BL / 6N male mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 0 . 5 h as shown in Figure 1N . Relative acetate and acetate m + 3 levels in mouse liver tissues ( M ) and mouse serum ( N ) were examined . ( O ) Relative acetyl - CoA and acetyl - CoA m + 3 levels in mouse liver tissues were measured .

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

Figure 4H&E (hematoxylin-eosin) staining (C, top) and oil red O staining (C, bottom) were examined.Relative mRNA levels of IL-6, were analyzed. were examined. Data in are from n=5 mice per group.Relative mRNA levels of , IL-1β, TNF-α, Cxcl1, Cxcl2, Col1a1 were analyzed. Data in are from n=5 mice per group.(J) Relative triglyceride level was examined. (K-L) Relative levels of serum ALT (K) and AST (L) were examined. Data in are from n=5 mice per group.(M-O) WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h as shown in Figure 1N. Relative acetate and acetate m+3 levels in mouse liver tissues (M) and mouse serum (N) were examined. (O) Relative acetyl-CoA and acetyl-CoA m+3 levels in mouse liver tissues were measured.


Figure 5 ( C - D ) p53 + / + and p53 - / - mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 1 h . Chromatin fractions of liver tissues were subjected to ChIP - seq assay using H3K9ac antibody ( left panel ) , H3K27ac antibody ( right panel ) . Heat map of H3K9ac / H3K27ac binding signal ( rows ) from - 1 kb to + 1 kb surrounding the centre of H3K9ac / H3K27ac - binding sites for ChIP - seq tags ( C ) . Heatmap of H3K9ac / H3K27ac binding signal ( rows ) from - 1 kb to + 1 kb surrounding the centre of H3K9ac / H3K27ac - binding sites for ChIP - seq tags of fatty acid metabolism related genes ( D ) . For ChIP - seq analysis , there were no technical replicates for anti - H3K9ac or anti - H3K27ac . ( F ) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53 − / − mice injected intraperitoneally with ethanol - d6 ( 2 g / kg ) or saline for 1 h . Bound SCD1 response elements ( RE ) were analyzed by qRT - PCR . Data in F are from n = 3 biological independent samples . WT , p53 − / − , ALDH2 − / − and p53 − / − ALDH2 − / − C57BL / 6N male mice were fed control diet and ethanol diet as shown in Fig . 4A . Relative mRNA level of SCD1 ( G , top panel ) was analyzed . Protein levels of p53 , ALDH2 , SCD1 , Histone H3 and Ac - H3 , caspase 3 , cleaved caspase 3 , p21 ( G , bottom panel ) in mice liver tissues were examined . Data in G are from n = 5 technical replicates from one of three independent experiments with similar results .

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

Figure 5(C-D) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 1 h. Chromatin fractions of liver tissues were subjected to ChIP-seq assay using H3K9ac antibody (left panel), H3K27ac antibody (right panel). Heat map of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags (C). Heatmap of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags of fatty acid metabolism related genes (D). For ChIP-seq analysis , there were no technical replicates for anti-H3K9ac or anti-H3K27ac.(F) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53−/− mice injected intraperitoneally with ethanol-d6 (2 g/kg) or saline for 1 h. Bound SCD1 response elements (RE) were analyzed by qRT-PCR. Data in F are from n=3 biological independent samples.WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were fed control diet and ethanol diet as shown in Fig.4A. Relative mRNA level of SCD1 (G, top panel) was analyzed. Protein levels of p53, ALDH2, SCD1, Histone H3 and Ac-H3, caspase 3, cleaved caspase 3, p21 (G, bottom panel) in mice liver tissues were examined. Data in G are from n=5 technical replicates from one of three independent experiments with similar results.


Figure 6WT and p53 − / − mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein . After 7 days , WT and p53 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . ( C ) Protein levels of p53 and SCD1 were examined by western blot . level were examined . WT and p53 − / − mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein . After 7 days , WT and p53 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . Relative hepatic triglyceride level ( D ) , relative serum ALT ( E , left panel ) and AST ( E , right panel ) level were examined . WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were injected with adeno - associated virus expressing AAV - GFP or AAV - SCD1 via tail vein . After 7 days , WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . ( H ) Protein levels of p53 and SCD1 are shown . WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were injected with adeno - associated virus expressing AAV - GFP or AAV - SCD1 via tail vein . After 7 days , WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . Relative hepatic triglyceride level ( I ) , relative serum ALT ( J , left panel ) and AST ( J , right panel ) were examined .

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

Figure 6WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (C) Protein levels of p53 and SCD1 were examined by western blot. level were examined.WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level(D), relative serum ALT (E, left panel) and AST (E, right panel) level were examined.WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (H) Protein levels of p53 and SCD1 are shown.WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level (I), relative serum ALT (J, left panel) and AST (J, right panel) were examined.


Figure 1A : Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines . Rhabdoid tumor cell lines ( red ) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin . These results confirm a previously published screen ( Osgood et al . , 2016 ) . B : Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines ( Teicher et al . , 2015 ) . The rhabdoid tumor cell line , G401 ( red ) , appears on the left side of the graph indicating this cell line is more sensitive to mithramycin . C : Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines . RT cell lines ( black ) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells ( grey ) . RT cell lines are not sensitive to three broadly - active chemotherapeutic agents : etoposide , doxorubicin or SN38 . Data represents mean with standard deviation derived from three independent experiments .

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

Figure 1A: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al., 2016).B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.