Fig. 3: CHD6 affects the DNA damage response through modulation of autophagy flux.

a Western blots (top) showing changes in ATG3 and LC3 levels in wild-type (wt) and monoallelic mutant iPSCs (mut) that were serum-starved (starv) and/or treated with chloroquine (chlo) or rapamycin (rapa); β-tubulin levels provide a loading control. Mean normalized band intensities from two experiments (±SD) were quantified and plotted relative to wt levels (bottom). b FACS profiles (left) of wild-type (wt) or monoallelic mutant iPSCs (mut) transfected with the GFP-LC3-RFP reporter. Plots (right) quantify the percent of RFP-only iPSCs over all RFP/GFP-double positive plus RFP-only cells in control and 2-h starvation conditions (mean ±SD). *: mean significantly different from control; P < 0.01, unpaired two-tailed Welsch t-test. c Representative images of wild-type (wt) or monoallelic mutant iPSCs (mut) immunostained for LC3 (green) and p62 (magenta) in the presence or absence of rapamycin from one experiment are shown. Box plots (mean with whiskers indicating 95th percentile intervals) quantify the number of puncta, mean signal and extent of LC3/p62 colocalization. *: mean significantly different from wt; P < 0.01, unpaired two-tailed Welsch t-test. Bar, 5 μM. d As in (c), but for γH2A.X levels (thick black lines indicate IQR, thin lines indicate 95% confidence intervals) after 30-min etoposide (ETO) treatment of wild-type (wt), heterozygous (het), or monoallelic mutant (mut) CMs. *: significantly different from wt; P < 0.01, Wilcoxon-Mann-Whitney test. Bar, 10 μM. e As in (d), but for γH2A.X (green) and CHD6 (magenta) after etoposide treatment of iPSCs. The line scan and colocalization index (ci) of the two signal profiles (bottom; γH2A.X foci indicated by arrows) exemplify the lack of signal overlap. Bar, 5 μM. f Comet assays and quantification of tails in wild-type (wt), heterozygous mutant (het), patient-derived (pat) or monoallelic mutant (mut) iPSCs treated with etoposide for 30 min and allowed 24 h to recover; numbers of cells analyzed (n) are indicated. *: significantly different from wt; P < 0.05, Wilcoxon-Mann-Whitney test. g As in (f), but for wild-type iPSCs treated with etoposide and allowed to 24 h recover in the presence or absence of autophagy inhibitors. *: significantly different from wt; P < 0.05, Wilcoxon-Mann-Whitney test. h Bar plots showing percentage (mean ±SD, n = 3 independent experiments) of wild-type (wt), heterozygous (het) or monoallelic mutant (mut) iPSCs that survived apoptosis, also in the presence or absence of autophagy inhibitors. *: mean significantly different from wt; P < 0.05, two-tailed unpaired Student’s t-test. i Bar plots showing the percentage (±SEM, n = 3 independent experiments) of wild-type (wt), heterozygous (het) or monoallelic mutant iPSCs (mut) in the G1, S, or G2 cell cycle phase upon 0, 10 or 30 min of etoposide treatment followed by 24 h recovery. *: significantly different from wt; P < 0.05, two-tailed unpaired Student’s t-test. j Significantly-enriched GO terms associated with the genes highlighted in (k). k Heatmap showing changes in mRNA levels (log₂) of genes differentially regulated in wild-type iPSCs upon 1 h of etoposide treatment. Those convergently up-/downregulated in monoallelic (mut) and heterozygous mutant (het) cells are highlighted (magenta/blue rectangles).