EMBO/SourceData huggingface dataset:
Figure 2(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.
Figure 3(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.
Figure 4(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.
Figure 5(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.
figf1(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.
figf2a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).(b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.
figf3(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM(b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.
figf4PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.
Figure 1A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.
Figure 2A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C
Figure 3A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.
Figure 4A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.
Figure 5C, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.H, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.
Figure 6B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.
Figure 7A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).
Figure 1B-F Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.G Percentages of ddaC clones showing severing defects at 16 h APF.H Quantitative analysis of unpruned dendrite lengths at 16 h APF.
Figure 2A Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.B Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.C Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.D-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.N, O Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae.O Protein expression of Msps and TACC in brain tissues. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.
Figure 3A-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.F-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.
Figure 4Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites.Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.I Percentages of ddaC neurons showing defective Kin-lacZ distribution.
Figure 5A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.
Figure 6A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.O Percentages of ddaC neurons showing severing defects at 16 h APF.P Quantitative analysis of unpruned dendrite lengths at 16 h APF.
Figure 7-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.G-J Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.
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).