Fig 2: Defective DNA Repair but Normal DNA Damage Sensing and Signaling in P1’s Cells(A–D) Survival of SV40 fibroblasts after IR (A), phleomycin (B), etoposide (C) and MMC (D) treatment. Results are expressed as the fraction of surviving cells in relation to untreated cells. Each point represents the mean value and standard deviation of three separate determinations. ATM- and Cernunnos-deficient cells were used as sensitive controls. Control-sensitive fibroblasts were from ATM-, Cernunnos-, and Fanconi-deficient patients.(E) Sensitivity of P1’s cells to phleomycin after WT-RAD50 or empty vector transduction. This experiment was performed three times. Control-sensitive fibroblasts were from a Cernunnos-deficient patient.(F) Functional complementation of phleomycin sensitivity provided by WT-RAD50 transduced into the patient’s cells compared with the empty vector. The selective growth advantage is scored as the increase in the index of mCherry-positive cells/mCherry-negative cells at various times compared with the initiation of the culture (index = 1). This experiment was performed twice.(G) Recruitment of GFP-NBS1 to DNA damage upon laser microirradiation in indicated cells. GFP-NLS (nuclear localization signal) is used as negative control. Irradiated zones are located in the red circles. Representative of three independent experiments.(H) Kinetics of recruitment of GFP-NBS1 and GFP-NLS to DNA damage in the control and the patient’s cells.(I) Immunofluorescence detection of P-KAP1 in primary fibroblasts before and 1 h after 0.5 Gy irradiation. ATM- and NBS1-deficient cells are used as negative controls. The image is representative of three independent experiments.(J) Phosphorylated forms of CHK2 and NBS1 detected by western blot analysis of whole-cell lysates from SV40 fibroblasts from a healthy control, an ATM-deficient patient, and P1 untreated or 1 h after 5 or 10 Gy irradiation. Vinculin immunoblot is used as loading control. An asterisk indicates a nonspecific band.(K) (Left) DNA content and histone H3 phosphorylation (P-H3) in G2 (pink rectangle) were analyzed by fluorescence-activated cell sorting (FACS). (Middle) FACS images of P-H3 in G2 in untreated and irradiated cells. (Right) G2/M checkpoint measured by the percentage of inhibition of entry into mitosis in cells after IR. Results represent the mean and SD from at least three independent experiments. Statistical significances are noted.

Fig 3: Deletion of PP4 by CD23/cre reduces CSR efficiency. a Immunoblots to detect p-ATR S431, p-Chk1 S345, p-NBS1 S343, NBS1, ?H2AX, H2AX, PP4, GAPDH and p84 in the cytosolic and nuclear fractions of B cells of the indicated genotypes that were left unstimulated (0), or stimulated with LPS + IL-4 for 24 h or 48 h. Data are representative of two independent trials. b Confocal microscopy images of immunofluorescent staining to detect RPA1 and quantitation of the intensity of RPA1 stainining in B cells of the indicated genotypes that were left untreated (resting) or stimulated with LPS + IL-4 for 24 h. c Confocal microscopy images of immunostaining to detect ?H2AX (red) and quantitation of ?H2AX-foci in B cells of the indicated genotypes that were left untreated (resting) or stimulated with LPS + IL-4 for 48 h. d Confocal microscopy images of immunostaining to detect NBS1 (green) and quantitation of NBS1-foci in the experiment described in c. e Confocal microscopy images of immunostaining to detect ?H2AX overlaid with NBS1 and quantitation of colocalized ?H2AX-NBS1 foci (index of correlation, Icorr) in the experiment described in c. Horizontal line = mean ± SD. Data from b–e are analyzed by MetaMorph. Values are for individual cells, and representative of two independent experiments. ns, not significant

Fig 4: ATR is an apical kinase in hypoosmotic stress-induced DDR. (A) G1-phase HeLa cells with knockout of histone H2AX or knockdowns of either ATM, ATR or Nbs1 were subjected to hypoosmotic stress for 1 h. WB was performed with an antibodies against the indicated DNA repair factors. Histone H3 was used as a loading control. The control (C) represents untreated HeLa cells. (B) Occupancy of DNA repair factors at the rDNA of HeLa cells with knockout of histone H2AX or knockdowns of ATM or ATR under hypoosmotic stress conditions (1 h). ChIP with antibodies against ATM, Nbs1, Mre11 and TopBP1 followed by qPCR using the rDNA amplicons positioned as indicated on the scheme in Figure 3B (A1: promoter, A2: 5'-28S rRNA region, A3: 3'-28S rRNA region; the amplicon from GAPDH gene was used as a negative control). Data are represented relative to the input. Values are means ± SD from at least three independent replicates.

Fig 5: Hypoosmotic stress-induced DDR leads to ATR/ATM-dependent silencing of rDNA transcription. (A) HeLa cells, untreated or exposed to hypoosmotic stress for 3 h, were simultaneously pulsed with 5-fluorouridine (FU) for 3 h. Throughout the figure, FU was revealed by immunocytochemistry. The DNA was stained with DAPI (blue). Scale bar: 15 µm. (B) HeLa cells, untreated or exposed to hypoosmotic stress for 0.5, 1 or 3 h, were pulsed with FU for 30 min. Box plots show the FU fluorescence intensities. Horizontal lines represent the median. (C) Quantification of FU fluorescence intensities in HeLa cells that were pre-treated with specific inhibitors of either ATM (KU55933), ATR (VE821) or DNA-PKcs (NU7026) and then subjected to hypoosmotic stress for 3 h. Horizontal lines represent the median. ***P < 0.0001, *not significant (unpaired t-test, n > 500). (D) Quantification of FU fluorescence intensities in HeLa cells with CRISPR/Cas9-based knockout of histone H2AX or RNA interference-based knockdowns of either ATM, ATR, DNA-PKcs (PRKDC) or Nbs1 that were subjected to hypoosmotic stress for 3 h. Horizontal lines represent the median. ***P < 0.0001, n.s.—not significant (unpaired t-test, n > 500). (E) qRT-PCR showing levels of pre-rRNA normalized to GAPDH mRNA in HeLa cells treated as in D. HeLa cells treated with Pol I inhibitor ACD were used as a negative control. The data are represented as the mean ±SD. ***P < 0.0001, n.s.—not significant (unpaired t-test, n > 500). (F) HeLa cells expressing homing endonuclease I-PpoI were mock-treated (-4-OHT) or treated with 4-hydroxytamoxifen (+4-OHT) for 16 to activate I-PpoI and stained for ?H2AX (green) and nucleolin (red). The DNA was stained with DAPI (blue). Scale bar: 20 µm. (G) HeLa cells expressing homing endonuclease I-PpoI were pre-treated with specific inhibitors of either ATM (KU55933), ATR (VE821) or DNA-PKcs (NU7026), and then treated with 4-hydroxytamoxifen (4-OHT) for 4 h to activate I-PpoI and pulsed with FU for 3 h. HeLa cells expressing I-PpoI, mock-treated (-4-OHT) or incubated with 4-OHT (C), were used as controls. Box plots show the FU fluorescence intensities. Horizontal lines represent the median. ***P < 0.0001, *not significant (unpaired t-test, n > 500).