Fig 2: UA induces DDR in embryonic CSCs. (a) The images of a comet assay from fluorescence microscopy at 10x and 40x magnification showing the fragmented DNA migration from the nucleoid body that forms a comet tail in NTERA-2 and NCCIT cells after 24 h treatment with 20 μM UA. (b) Graphical representation of comet length was analyzed as the fold change versus the control and percentage of comet-positive cells with UA treatment in embryonic CSCs. ∗∗∗p < 0.001 and ∗∗p < 0.01 (Student's t-test). (c) Immunoblotting analysis of NTERA-2 and NCCIT cells after treatment with 10 and 20 μM UA for 24 h showing manifestations of phospho-histone BRCA1, ATM, ATR, CHK1, and CHK2 proteins. Exposure rates were measured by densitometry and were standardized in β-actin levels. Data were received from three different experiments.

Fig 3: CDS-3078 induces cell cycle arrest at the G2/M phase. (A) HeLa cells were pretreated with 2, 5 and 10 µM of CDS-3078 for 24 h, stained with PI and then subjected to DNA content analysis using flow cytometry. Data represents three independent repeats. (B) Quantification of the percentage of G0/G1, S and G2/M HeLa cells at the various administration times. (C) HeLa cells were treated with CDS-3078 (5 µM) for 4, 8 and 16 h. The expression levels of the proteins associated with the cell cycle were examined using immunoblotting to detect expression levels of CDK1, cyclin B1, CDC25C, CHK1, p-CHK1, CHK2, p-CHK2, ATR, p-ATR, H2AX and γ-H2AX. (D) The relative density was determined using grayscale analysis on ImageJ 1.8.0_112 (National Institutes of Health) normalized to corresponding non-phosphorylated total protein. CDS-3078, 2-[1-(4-(Benzyloxy)phenyl)-3-oxoisoindolin-2-yl)-2-(4-methoxyphenyl)acetic acid; CHK, checkpoint kinase; CDC25C, M-phase phosphatase 3; H2AX, H2A histone family, member X; ATR, ataxia telangiectasia and Rad3-related protein; p-, phosphorylated. *P<0.05 vs. control.

Fig 4: M3814 over-activates IR-induced p53 apoptotic signaling in acute leukemia cells. (A) Molm-13 cells were pre-treated with M3814 (1 µM) or vehicle for 45 min before exposure to IR (2 Gy). Protein lysates were isolated 4 h after irradiation and analyzed by MSD for phospho-ATM (Ser1981) and ATM. Results shown as fold change in p-ATM normalized to total ATM protein. (B) Molm-13 cells were treated as in A. Protein lysates were isolated 4 h after irradiation and analyzed by Western blotting using antibodies for phospho-Kap1(Ser824), Kap1, phospho-CHK2(Thr68), CHK2, phospho-p53(Ser15), p53, Puma, cleaved caspase-3 and GAPDH. Panel displays cropped images from repeat blots divided and probed for different targets. Full-sized blots are presented in Supplementary Fig. 1. (C) Molm-13, Molt-4, MV4-11, THP-1 and HL-60 cells were M3814-treated and irradiated as in A. RNA was isolated 4 h post-IR and p53 target gene expression was assessed by qPCR using probes for p21, MDM2 and Puma. Expression levels were normalized to GAPDH. (D) Molm-13, Molt-4, MV4-11, THP-1 and HL-60 cells were pre-treated with increasing M3814 concentrations for 45 min before exposure to IR (1 Gy or 2 Gy). Cell growth/viability was assessed via CellTiter Glo assay on d 4. Shown are individual M3814 dose–response curves (top) and overlays of Bliss synergy on relative viability landscapes (bottom). Bliss synergy was determined using Combenefit software. (E) Molm-13, Molt-4, THP-1 and HL-60 cells were treated as in A, then exposed to 1 Gy IR and 24 h later cells undergoing apoptosis were assessed by flow cytometry using a PE-conjugated annexin V. *p < 0.05, **p < 0.01, ***p < 0.001. NS: p > 0.05.

Fig 5: PRR14 inhibits CHEK2.CHEK2 mRNA expression (a), CHEK2 protein expression (b) and p-CHEK2 (T68) protein expression (c) in PRR14 genetically unaltered and altered (amplified and mutated) breast cancer cases in TCGA database are statistically analyzed by two-tailed Student’s t-test. CHEK2 protein expression is detected by immunostaining in xenografts in nude mice from established MCF7 cell lines and MDA-MB-231 cell lines (d), as well as human breast cancer (e). CHEK2 transcription in human breast cancer is also detected by qRT-PCR (f). The data are quantified and two-tailed Student’s t-test is employed to determine the significance of the difference. Established MCF7 and MDA-MB-231 (g) PRR14-overexpressing and control cell lines are treated with various of genotoxic chemicals including Bleo, Eto, 5-FU, H2O2 and HU at indicated concentrations for indicated time. Key components of the ATM/CHEK2/P53 signaling pathway are detected by immunostaining. And CHEK2 protein expression (h) and mRNA expression (i) are detected by immunostaining and qRT-PCR, respectively. The data are analyzed by two-tailed Student’s t-test. Established MCF7 and MDA-MB-231 (j) PRR14-overexpressing and control cell lines are treated with Eto at indicated concentration for indicated time to induce p-CHEK2 (T68), which is detected by immunostaining and quantified and normalized by CHEK2 total protein (k). The data are analyzed by two-tailed Student’s t-test.