Fig. 7: PARPi triggers an S-phase specific accumulation of γH2AX and RAD51 in CHD6-deleted cells, which display highly functional DNA double strand break repair. | Nature Communications

Fig. 7: PARPi triggers an S-phase specific accumulation of γH2AX and RAD51 in CHD6-deleted cells, which display highly functional DNA double strand break repair.

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

Fig. 7

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. All experiments are n = 3, centre lines of all foci data graphs are arithmetic means ± CI95%. Source data are provided as a Source Data file. Panel a Logarithmically dividing WT and ΔCHD6 cells were treated with 2.5 µM PARPi or DMSO for 1h and immunostained for γH2AX and DAPI. Images are representative of cells in mid S-phase based on relative DAPI signal. Scale bars = 5 µm. Panel b For the experiment described in (a), γH2AX foci per nucleus for cells in either G1 or mid S-Phase were enumerated. Panel c For the experiment described in (a), refined γH2AX signal (that accounts for γH2AX focal intensity, focal number, and nuclear volume) in either G1 or mid S-Phase was measured. Panel d S-phase cells from (b) were immunostained for γH2AX and RAD51, and enumerated for the arithmetic mean number of RAD51 foci per nucleus; n = 3. Representative images are from PARPi-treated ΔCHD6 S-phase cells. Scale bars = 5 µm. Panel e Logarithmically dividing ΔCHD6 RPE-1 cells were incubated in media with 2.5 mM PARPi or an equal volume of DMSO for 2h, then placed into fresh media and harvested immediately (0h), or after another 1, 2, and 3h. Cells were immunostained for γH2AX and RAD51, and enumerated as in (b, d). Panel f Logarithmically dividing WT and ΔCHD6 RPE-1 cells were irradiated with 2 Gy gamma ray ionizing radiation (IR), fixed between 0.5-24h later, and immunostained for γH2AX and DAPI. Graphs are γH2AX foci per nucleus for cells predominantly in (left to right) G1-phase, S-phase, or G2-phase. Panel g Logarithmically dividing WT and ΔCHD6 cells were treated with 50 µM H2O2 (or PBS), detergent extracted, immunostained for PARP-1, imaged and quantified for nuclear PARP-1 signal; n = 3.

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