Fig 2: Failure of maintenance of nocodazole-induced G2 arrest and impaired DNA damage–induced apoptosis in CHEK2KO cells.(A) Kinetics of cell cycle progression of CHEK2WT and CHEK2KO cells after nocodazole treatment (2 µM) for 0, 6, and 12 hours. (B) The percentage of cells that arrest at G2 after nocodazole treatment at different time points and concentrations. Data are expressed as mean ± SEM (n = 3). *P < 0.05, nonparametric Mann–Whitney U test. (C) Cellular apoptosis analysis by flow cytometry in CHEK2WT and CHEK2KO cells treated with different concentrations of Adriamycin after 24 hours. (D) The percentage of apoptotic cells calculated by flow cytometry from C. Data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, nonparametric Mann–Whitney test. (E) Cell viability detected by a crystal violet assay after cells were treated with different concentrations of nocodazole or Adriamycin. (F) Normalized cell population/well calculated by ImageJ software (NIH) from E. Data are expressed as mean ± SEM (n = 3). *P < 0.05, nonparametric Mann–Whitney U test. (G) Protein levels of pan-p53, phosphorylated p53 (serine 20), and Chk2 in CHEK2WT and CHEK2KO cells after Adriamycin treatment for the indicated times.

Fig 3: Dsg3 depletion causes further induction of p53 expression and activity in response to stress signals.a Western blotting of siRNA pre-treated NTERT cells with and without UVB irradiation for the indicated proteins with the quantitation shown on the right (n = 3 biologically independent samples, *p < 0.05). b Western blotting for p53/p21WAF1/CIP1 in cells treated with or without actinomycin D (Act D, 5 nM) and mitomycin C (MMC, 5 µg/ml) for 24 h, respectively. The quantitation data are shown on the right. Enhanced expression of p53/p21WAF1/CIP1 was shown in cells with Dsg3 knockdown and treated with drugs. c Mechanical stretching induced increased expression of p53 and p21WAF1/CIP1/ Bax in Dsg3 KD cells. The siRNA pre-treated cells were seeded at confluent density in BioFlex plates and then subjected to cyclic strain (TX-5000, 20% amplitude, 1 Hz) for 4 h the following day. Lysates were extracted either immediately after strain or 2 h and 24 h later, respectively, after transferring to a stationary state, along with static control cells. d Western blotting analysis for PCNA and cyclin A in siRNA treated cells with and without UV. e Western blots for the indicated proteins upstream of p53 as well as phospho-p53-S20 in siRNA pre-treated cells with and without UVB (30 mJ/cm2). f Qnatitation of 53BP1 nuclear staining (n = 10, mean ± S.D., **p < 0.01, ***p < 0.001). g Western blotting in siRNA transfected cells treated in the presence and absence of ATM inhibitor KU55933 (20 µM) and p38 MAPK inhibitor SB203580 (20 µM), respectively. All cells were exposed to UVB (30 mJ/cm2) in this case. Cells were treated with drugs 1 h before UV and were grown overnight before lysate extraction. h The expression of p53 in the same samples as phosphorylated ATM and CHK2 (the last two corrected for total protein) following the indicated drug treatments in Dsg3-depleted cells. The data are expressed as the band intensities in the Dsg3-depleted cells relative to the corresponding scrambled siRNA controls, which were normalized to 1

Fig 4: CRISPR/Cas9-mediated CHEK2 gene inactivation in CRC cell lines.(A) CRISPR-directed CHEK2 inactivation was designed targeting exon 10. (B) The aberrant sequence signal after the expected break site of the edited cell pool by Sanger sequencing (red arrowhead). (C) Chk2 protein level of the selected CHEK2KO clones assessed by Western blot. (D) Proliferation of CHEK2WT and CHEK2KO cells detected by CCK8 after cultured for the indicated time. Data are expressed as mean ± SEM (n = 6), nonparametric Mann–Whitney U test.

Fig 5: ITGB1 expression correlates with the expression of proteins related to the ATM/CHK2/CDC25c pathway. A. The relationship between ITGB1 expression and that of 34 genes in the DNA-DSB response pathways. B-D. Protein levels of ATM (p-ATM), CHK2 (p-CHK2), and CDC25c (p-CDC25c) were detected by western blotting in shITGB1 and ITGB1 overexpression groups.