Fig 2: Effect of ATR inhibition on fludarabine- and UV-C -induced cytotoxicity in p53-defective CLL cells(A) 17p-deleted CLL samples (CLL12, 23 and 40), containing ≥ 71 % of cells with deletion, were incubated for 96 h with increasing concentration of F-Ara-A in the presence or absence of 10 μM VE-821. (B) One of these samples (CLL12) was also irradiated with increasing UV-C doses and incubated for 24 h in the presence or absence of 10 μM VE-821. Cell survival was measured by the MTT assay.

Fig 3: Knockdown of ATM, ATR, and DNA-PKcs decreases AAV2 replication. HEK293T cells were transduced with lentiviral vectors expressing kinase-targeted shRNA or scramble shRNA, as indicated, prior to pHelper transfection and AAV2 infection. (A) Western blot analysis. At 2 dpi, the cells were collected and lysed for Western blotting using anti-p-ATM(S1981), anti-p-ATR(T1989), and anti-p-DNA-PKcs(S2056) antibodies, respectively. ß-actin was used as a loading control. (B and C) Southern blotting. At 2 dpi, the cells were used to extract Hirt DNA for Southern blotting. The blot was probed with an AAV2 repcap probe and a mtDNA probe, respectively. (B) A representative blot is shown. dRF, double replicative form; mRF, monomer replicative form; and ssDNA, single-stranded DNA. AAV2 DNA recovered from PvuII-digested pSSV9 was used as a size maker (M) at 4.7 kb. Probed mtDNA served as a loading control. (C) Quantification of mRF DNA. The data were normalized to the amount of mtDNA and represented on the results of 3 independent experiments. P values were calculated by using Student's t test (**, P < 0.01; ***, P < 0.001; n.s., no statistically significant difference).

Fig 4: The ATR-CHK1 signaling is a critical barrier protecting cells from RPA exhaustion and replication catastrophe in cells challenged with SN-38. (A) WT cells were pulsed with BrdU after (left) or before (right) drug inductions (5 h; 40 nM SN-38 ± 2 µM VE-822). Doublets and debris were excluded and 20,000 cells were analyzed per sample. (B) Immunofluorescence analysis of WT cells challenged with drugs (same parameters). Target proteins were immunodetected, DNA was counterstained with DAPI and cells were observed with a Zeiss Axio Imager (apotome, × 63 objective) scale bars: 20 µm. (C) Flow cytometry analysis of WT cells challenged with drugs (same parameters). DAPI was used to stain DNA. 20,000 cells were analyzed per sample. ssDNA, RPA32, RPA32pS4-8 and ?H2AX positive cells were arbitrary gated on the SN-38 + VE-822 quadrants and gates were pasted all conditions. (D) ?H2AX and RPA32 were detected in immunofluorescence (upper panel). DAPI was used to counterstain DNA. Cells were observed with a Zeiss Axio Imager with apotome (× 63), scale bar: 20 µm or analyzed in FACS. Representative images from 3 independents experiments are shown. A representative cell of each sample is magnified (white arrow), without DAPI. Cross sections show the relative levels of RPA32 and ?H2AX (“Profile” Zen Tool). For flow cytometry, RPA32 and ?H2AX were detected, gates were set on the SN-38 quadrant and pasted on the other conditions. The increase of ?H2AX intensity between SN-38 and SN-38 + VE-822 is highlighted in the orange gates. Percentages of double positive (orange gates) were plotted. The experiment was repeated 3 times. Mann–Whitney test was applied. (E) WT, MUT and REV cells were analyzed by flow cytometry. ?H2AX and RPA32 double positive cells were gated (orange gates). The experiment was repeated 3 times. Mann–Whitney test was applied. (F) Flow cytometry analysis of WT cells challenged with SN-38 + VE-822 with or without roscovitine (ROSC, 20 µM). ?H2AX and RPA32 were detected. Double-positive cells were gated (Orange) and plotted. Mann–Whitney test was applied.

Fig 5: Effects of Claspin or ATR depletion on Chk1 activation by various biological stresses and on the factors involved in growth-related pathways. (A) Claspin(f/-) MEF cells were treated with Ad-Cre or nontreated and exposed to various stresses for 3 h. (B,D) HeLa cells were transfected with siRNA for Claspin (B) or for ATR (C), and were exposed to indicated stresses for 3 h before the harvest. The whole cell extracts were analyzed by Western blotting with antibodies indicated. SAPK/JUNK and p38 MAPK (MAPK activated by stresses and growth factors); ERK1/2 (MAPK activated by growth factors and mitogen); 4EBP1 (phosphorylated by mTOR and required for activation of translation); PDK1 (required for activation of mTOR); Mcm2 (phosphorylated by Cdc7 and required for replication); Mcl-1 (anti-apoptotic factor). Phosphorylated forms represent activated states. -, control siRNA. (D) Extracts prepared from HeLa cells treated with the stresses indicated were incubated in the absence of presence of λppase, and analyzed on a low concentration gel.