Fig 2: ATR inhibition leaves ATM signaling intact. (A) IF of ATM-pS1981 (green) and 53BP1 (red) foci in 82-6 hTert cells 1 h after exposure to 1 Gy. Blue: DAPI staining. (B) IF analysis of ATM-pS1981 foci (green) in late-S/G2 (Cyclin B1 positive, red) 82-6 hTert cells exposed to 1 Gy, 1 h after treatment with the indicated concentrations of ATRi. (C) Western blot analysis of 82-6 hTert cells treated with ATRi or ATMi for 1 h and exposed to 0 or 4 Gy under continuous incubation with the inhibitors. GAPDH serves as a loading control. (D,E) Western blot analysis monitoring DNA-PKcs, ATM, ATRIP and Ku70, as well as activation of the downstream PIKK targets p53-pS15, KAP1-pS824, Chk2-pT68 and Chk1-pS345 in 82-6 hTert cells treated with ATRi and ATMi after exposure to 4 Gy. GAPDH, as well as non-phosphorylated Chk1 and Chk2 serve as loading controls. (F) IF of ATM-pS1981 (green) and Chk2-pT68 (red) 30 min after exposure to 2 Gy of the Seckel syndrome cell lines, F02-98 and DK0064, as well as of GM847-ATRkd cells tested in the absence (-DOX) or presence (+DOX) of DOX.

Fig 3: ATR is essential for the manifestation of the G2-checkpoint in S-phase irradiated cells. (A) Percentage of 82-6 hTert cells in G2-phase of the cell cycle as determined by single parameter flow cytometry as a function of time after exposure to 0 Gy (NI: non-irradiated) or 2 Gy of IR (X-rays). DNA content was determined by staining with propidium iodide (PI). Results are shown for untreated cells (untr), as well as for cells incubated with the indicated inhibitors. (B) As in (A) for A549 cells exposed to 4 Gy of IR. (C) A549 cells were pulse labeled for 30 min with BrdU and were exposed to 4 Gy of IR. The percent of BrdU positive (BrdU+) cells in G2-phase was measured as a function of time thereafter using two parameter flow cytometry. Results are shown for NI and untr cells, as well as for cells incubated with ATRi. (D) Western blot analysis of GM847-ATRkd cells, showing ATRkd expression after 48 h incubation with 3 µg/ml doxycycline (+DOX), or without doxycycline (-DOX). KU70 and RPA32 proteins serve as loading controls. Original, non-cropped membranes are shown in Fig. S4A. (E) Percentage of G2-phase, GM847-ATRkd cells irradiated with 4 Gy and analyzed in the absence of doxycycline (-DOX). (F) As in (E) for cells tested in the presence of doxycycline (+DOX). All data points represent the means and standard deviations (only visible when are larger than the symbol) estimated from three independent experiments.

Fig 4: DNA-PKcs deficiency results in hyperactivation of the G2-checkpoint in cells irradiated during the S-phase of the cell cycle. (A) As in Fig. 1A for the DNA-PKcs deficient cell line, M059J, exposed to 2 Gy and treated with indicated inhibitors. (B) As in (A) for DNA-PKcs proficient M059K cells. (C) As in Fig. 1A for 82-6 hTert cells exposed to 2 Gy and treated with the indicated inhibitors. (D) As in (A) for M059J cells, irradiated with 10 Gy and treated with the indicated inhibitors. All data points represent the means and standard deviations (only visible when are larger than the symbol) estimated from three independent experiments. (E) Western blot analysis of pCHK1-S345, a marker of ATR activation, after treatment with the indicated inhibitors of parental A549 (A549wt) and DNA-PKcs knock-out, A549 (A549DNA-PKcs-/-) cells, enriched in S-phase by a single thymidine block, exposed to 10 Gy of IR and collected for analysis 9 h after irradiation. The levels of DNA-PKcs confirm the successful knockout, while the levels of CHK1 and KU70 serve as loading controls. (F) As in (E) for ATM deficient AT hTert cells analyzed 3 h after exposure to 10 Gy IR. CHK1 and RPA32 serve as loading controls. Original non-cropped images of the scanned western blot membranes are shown in Figs S4B,C.