Fig 2: The CHK1-PLK1 axis modulates DSB repair by targeting RAD51. (A) The role of CHK1 and PLK1 on HR. DR-U2OS cells were infected with I-SceI for 36 h, before treatment with UCN-01 (10 nM), BI2536 (10 nM) or both for 12 h. The percentage of GFP- positive cells is shown. (B) The role of CHK1 and PLK1 on RAD51 focus formation. U2OS cells were synchronized at late S phase, pretreated with UCN-01 (10 nM), BI2536 (10 nM), or both for 2 h, followed by CPT (2 μM) treatment for 1 h and immunofluorescence to detect RAD51 foci in γ-H2AX-positive cells. (C) The effect of combined gallotannin and UCN-01 treatment on HR repair. DR-U2OS cells were infected with I-SceI for 36 h, then treated with UCN-01 (10 nM), gallotannin (10 μM), or both. The percentage of GFP-positive cells is shown. (D) The effect of combined gallotannin and UCN-01 treatment on chromosome aberrations. HeLa cells were pretreated with UCN-01 (10 nM), gallotannin (10 μM), or both for 12 h, X-ray irradiated (5 Gy) and then treated with colchicines (0.4 μg/ml) for 6 h before analysis by chromosome spread assay. Representative images are shown. Blue arrows (others), dicentric, deletion, ring; green arrows, fusions; red arrows, breaks. (E) The percentage of chromosome aberrations in part D. More than 100 mitotic chromosomes were randomly analyzed. (F) The effect of combined gallotannin and UCN-01 treatment on sensitizing HeLa cells to IR treatment. HeLa cells were treated with UCN-01(10 nM), X-ray irradiation (2 Gy) or both, in the presence of an increasing amount of gallotannin (0, 1, 5, 10 μM) for 2 weeks. (G) The effects of inhibiting RAD51 expression on HR-mediated DSB repair in U2OS cells. DR-U2OS cells stably expressing the indicated constructs were first infected with I-SceI before siRNA transfection against RAD51 (3′UTR). The repair efficiency was analyzed as in part A. (H) Quantitation of chromosome aberrations upon RAD51 down regulation in HeLa cells. HeLa cells stably expressing the indicated constructs or RAD51 siRNA (3′UTR) were irradiated (5 Gy) before analysis by chromosome spread assay as in part D. More than 100 mitotic chromosomes were randomly analyzed. (I) Relative cell survival in HeLa cells. HeLa cells stably expressing the indicated constructs were treated with RAD51 siRNA (3′UTR), irradiated (0, 1, 2, 5 Gy) and the cell survival was determined. All data are derived from three independent experiments. *P < 0.05; ** P < 0.01; ns, not significant.

Fig 3: Proposed mechanism of activation of DDR through interaction of PLAUR and TLR4. Damage molecule serving as a ligand of PLAUR/TLR4 receptor complex is released from the cell nucleus. PLAUR/TLR4 auto-/paracrine signaling promotes CHK1 phosphorylation, activation and nuclear import of RAD51. Downregulation of PLAUR in TP53 WT cells results in delayed DNA repair and TP53-dependent cell cycle arrest and cell death. Cell cycle arrest in TP53-mutated PLAURsi cells is compromised and results in abnormal mitosis

Fig 4: Immunohistochemistry detection mapped the expression of signaling molecules relative to the ATM/ATR pathway in chordoma tissues and adjacent normal tissues stained with hematoxylin. p-ATM was moderately expressed in the nucleus and cytoplasm of adjacent normal cells, whereas it was highly expressed and largely accumulated in the nuclei of chordoma cells. p-ATR was expressed at a low level in adjacent normal cells; however, it was highly expressed in the nuclei and cytoplasm of chordoma cells. A small increase in γ-H2AX expression was observed in the nuclei of chordoma cells compared with adjacent normal tissues. No obvious difference in BRCA2 expression was observed between chordoma and adjacent normal cells. RAD51 expression was low in normal cells, whereas RAD51 staining was widely and substantially distributed in the nuclei and cytoplasm of chordomas. ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3 related; p, phosphorylated; γ-H2AX, phosphorylated H2A histone family, member X; BRCA2, breast cancer susceptibility gene 2. Magnification, ×400.

Fig 5: ATM/ATR signaling in chordoma cells exposed to low-dose radiation. In U-CH1 cells, 1 and 2 Gy radiation led to an upregulation in the expression of p-ATM, γ-H2AX and RAD51 proteins, but not p-ATR and BRCA2. p-ATM, p-ATR and RAD51 were upregulated in U-CH2 cells compared with Con cells, whereas γ-H2AX expression level increased only marginally and BRCA2 remained unchanged. *P<0.05 vs. Con. ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3 related; p, phosphorylated; γ-H2AX, phosphorylated H2A histone family, member X; BRCA2, breast cancer susceptibility gene 2; Con, untreated control cells.