Loss of CCDC6 affects H2AX phosphorylation after DSBs.

<p>(<b>a</b>) In the WCL of two representative CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL), thirty minutes after 1–5 Gy IR exposure, the phosphorylation of H2AX was detected with the mouse anti-pH2AX S139 by western blot. Anti-total H2AX was used as a loading control. The immunoblots with anti-CCDC6 and α-tubulin antibodies were shown in the bottom. (<b>b</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. (<b>c</b>) Immunofluorescence analysis of pH2AX S139 foci in CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL), thirty minutes after 1, and 5 Gy IR exposure. Nuclei were counterstained with DAPI. Magnification_ was at 63x. (<b>d</b>) Quantification of pH2AX S139 foci number. At least 300 cells were analysed per experiment. Error bars indicate the standard mean error. (<b>e)</b> CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL) transfected with expression vectors encoding CCDC6wt, CCDC6T434A or the empty vector were treated with etoposide at 1, 2,5 and 5 µM for 8 h and western blot analysis of pH2AX S139 and myc-tagged proteins were performed. (<b>f</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. The anti-pSer-1981-ATM and the ATM hybridization are shown at bottom of the figure.