Fig 2: ATM and ATR are both essential for the manifestation of checkpoint in G2-cells exposed to low IR doses. (A) Normalized mitotic index (MI) calculated from bivariate PI, histone H3-pS10 flow cytometry in 82-6 hTert cells; untreated (untr) or treated with indicated PIKK inhibitors. The values of MI used for normalization in different experiments were: MIuntr = (2.75–3.05%), MIATRi = (3.76–5.62%), MIATMi = (3.19–6.15%). Mainly cells irradiated in G2 reach M-phase within the observation time. (B) Western blot analysis of GM847-ATRkd cells, showing ATRkd expression 48 h after incubation with 3 µg/ml doxycycline (DOX). Bands corresponding to ATR and ATRkd are indicated. The signals of Ku70 and Rpa32 serve as loading controls. (C) Normalized MI as function of time after exposure to 4 Gy of DOX-treated (+DOX) GM847-ATRkd cells. The response of cells analyzed in the absence of DOX (-DOX) are also shown for comparison. (D) Western blot analysis of ATR and ATRIP in 82–6 hTert cells, 48 h after mock-transfection or transfection with siRNA targeting ATR. GAPDH serves as a loading control. (E) Normalized MI as a function of time after exposure to 2 Gy of 82-6 hTert cells transfected with siATR 48 h prior to irradiation. Shown are results of one representative experiment. (F) Normalized MI as a function of time after exposure of AT5Biva cells to 2 Gy, in the presence of ATMi or ATRi. The values of MI used for normalization in different experiments were: MIuntr = (2.52–3.04%), MIATRi = (2.78–4.02), MIATMi = (2.44–3.18%). Other details as in (A). (G) Normalized MI as a function of time after exposure to 2 Gy of 82-6 hTert cells, treated with Chk1 or Chk2 inhibitors as indicated. Dotted lines show for comparison results obtained in the absence of any treatment (untr) or after treatment with ATRi. Unless otherwise stated, all data points show the mean and standard deviation (visible only when larger than the symbol) calculated from three independent experiments.