Fig. 1: CHD6 loss or introduction of a catalytic site mutation sensitizes cells to PARP1/2-trapping inhibitors and/or depletion of BRCA1. | Nature Communications

Fig. 1: CHD6 loss or introduction of a catalytic site mutation sensitizes cells to PARP1/2-trapping inhibitors and/or depletion of BRCA1.

From: CHD6 has poly(ADP-ribose)- and DNA-binding domains and regulates PARP1/2-trapping inhibitor sensitivity via abasic site repair

Fig. 1

For all statistical analysis in this figure, 2-way ANOVAs followed by Tukey post hoc tests were carried out. ns = not significant (p > 0.05), *p < 0.05. ** p < 0.01. ***p < 0.001.****p < 0.0001. Source data are provided as a Source Data file. All clonogenic survival assay line graphs represent data normalized to untreated wildtype controls as arithmetic means ± SEM. In all cases, wildtype data are indicated in blue, while CHD6 negative data are in green. Panel a RPE1 were subjected to CRISPR/Cas9-mediated CHD6 gene deletion (ΔCHD6) and immunoblotted for indicated proteins. Panel b WT and ΔCHD6 cells were treated with increasing peroxide (left) or 50 µM H2O2 in PBS and/or increasing doses of the PARPi Olaparib in media (right), then scored for colony formation 7 days later. All data is normalized relative to DMSO alone, n = 6. Panel c WT and ΔCHD6 RPE-1 cells were plated and treated with increasing PARPi (left) or 2.5 nM PARPi and increasing H2O2. All data is normalized relative to DMSO alone; n = 6. Panel d Sequencing outcomes of successful CRISPR base editing of endogenous CHD6 (in RPE-1 cells) to introduce K492G + T493A = Catalytic Dead = “CHD6CatDead”. Panel e Immunoblot of CHD6 and loading control KAP-1 in RPE-1 expressing WT CHD6 and mutant from (d). Panel f WT and CHD6CatDead-expressing cells were treated with increasing PARPi then scored for colony formation 7 days later; n = 3. Panel g WT and ΔCHD6 RPE-1 cells were transfected with siRNA against BRCA1 or a scrambled control (siMock) and, 3 days later, treated with PARPi as in (d) and scored for colony formation; n = 3. Data in line graph is normalized relative to DMSO alone (with **** reflecting 2-way ANOVA comparisons of all lines relative to one another). Panel h Bar charts of resting state normalized to the WT RPE-1 transfected with siMOCK; error bars represent SEM, with statistical analysis being Mann-Whitney t-tests. n = 4. Immunoblot of BRCA1 is shown below to confirm siRNA efficacy.

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