FIGURE 3. GABA_AR activation regulates S-phase cell cycle progression by activation of PIKK and phosphorylation of histone H2AX.

a, Immunostaining of ES cells for -H2AX after 6 h of exposure to muscimol. Open arrow, -H2AX⁺ colony; filled arrow, baseline reactivity. b, Punctate nuclear localization of -H2AX staining in a single ES cell. c, Single ES cells filled with a gap-junction-permeable dye to reveal electrochemical coupling between cells within colonies; arrows indicate injected cells (n = 6). d, Western blot (WB) analysis of -H2AX in ES cells after exposure to muscimol or bicuculline and ATM/DNA-PK inhibitor (A/D i) for 6 h. e, Comet assay for DNA damage by 2 h of exposure to muscimol in ES cells. f, Quantification of e. g, Quantification of ATR RNAi-treated ES cells in S phase after 2 h of exposure to muscimol. h, i, Number of P-ATM^high cells by flow cytometric analysis after 2 h of exposure to muscimol in 3 subunit siRNA-treated cells. Dox., doxyrubicin. j, Cell cycle profiles of R1 ES cells with and without ATM/DNA-PK inhibitor (A/D i). k, ES cell numbers at 48 h with control siRNA or H2AX and control or H2AX and GABA_AR 3 subunit siRNA (n = 3). l, Effects of muscimol on H2AX siRNA-treated ES cells in a 2 -h cell doublet analysis (n = 3). m, Cell cycle profiles of R1 ES cells transfected with GABA_AR 3 and H2AX siRNAs. n, Flow cytometric analysis of ES cells after a 50-min BrdU pulse. BrdU increases with 3/H2AX siRNA. o, Flow cytometric analysis of R1 ES cells treated with the ATM/DNA-PK (A/D) inhibitor. BrdU increases in the presence of inhibitor. Error bars indicate s.e.m. (two-tailed Student's t-test). Scale bars, 200 m (a), 5 m (b), 50 m (c) and 100 m (e).