Fig 2: Effect of CHK1 siRNA knock down on CCC DNA formation during HBV infection in HepG2-NTCP cells. The HepG2-NTCP cells were transfected with the CHK1 siRNA (CHK1si) or negative-control siRNA (Controlsi). Subsequently, they were infected with HBV. Cells were harvested for analysis of CHK1 protein levels by Western blotting (A) and HBV PF DNA by Southern blotting (B) on day 2 and day 3 postinfection. ß-Actin was used as a loading control for Western blot analysis in panel A. HBV PF DNA was detected with a 32P-labeled HBV DNA probe in panel B. Quantitative results from multiple independent experiments were shown to the right. The data are expressed as CHK1 protein levels normalized to actin (A) or CCC DNA levels normalized to PF-RC DNA (B), with the normalized CHK1 protein or CCC DNA level from the mock-treated cells set to 1.0. *, P = 0.05; **, P = 0.01.

Fig 3: Western blotting of DDR signaling kinases, partners and substrates. (A–D) MCF7 Cells were transfected with CUX1-specific siRNA. Nuclear extract were prepared and analyzed by immunoblotting. (A) Cells were exposed to 10 Gy of IR, incubated for 1 h prior to harvest and the extracts were immunoblotted for CUX1 to assess knockdown. (B) Cells were treated as in (A) and immunoblotted for ATM, p-ATM, 53BP1, Chk2 and p-Chk2. Actin was used to control for equal loading. (C) Cells were exposed to 20 Js of UV, incubated for 2 h prior to harvest and the extracts were immunoblotted for ATR, Chk1 and p-Chk1. (D) Cells were exposed to 10 Gy of IR, incubated for 6 h prior to harvest and the extracts were immunoblotted for p53 and CUX1. Actin was used to control for equal loading. (E) Nuclear extracts from Cux1Z/Z and wild-type MEFs were immunoblotted for ATM or ATR following exposure to 10 Gy of IR (left panel) or 20 Js of UV (right panel), respectively.

Fig 4: DSB analysis of HCT116 and strains expressing wild type and mutant RPA2. Parental HCT116 and strains ectopically expressing wild type or mutant RPA2 were depleted of endogenous RPA2 by transfection of the 3' UTR siRNA before a 24 h treatment with 1 µM Gö6976 and 2 mM thymidine. Cells were then harvested for analysis of DSBs by pulsed field gel electrophoresis. RPA2 depletion triggers DSB formation in HCT116 but this is rescued by ectopic expression of mutant or wild type RPA2. Neither thymidine nor CHK1 inhibitors alone or in combination enhance DSB formation in RPA2 depleted cells ectopic expression of mutant RPA2s is still able to rescue HCT116 from DSB formation.

Fig 5: Model of synthetic lethality between miR-1193 inhibition and DNA-PKcs deficiency.a Interference with either DNA-PKcs or miR-1193 alone did not reduce the viability of cancer cells, while cell death was achieved by combined abolition of DNA-PKcs and miR-1193. b Function loss of DNA-PKcs resulted in dysregulation of the NHEJ-mediated DSB repair pathway (left panel). Inhibition of miR-1193 led to blockade of the FEN1-mediated HR/MMEJ repair pathway (middle panel). Combined abolition of DNA-PKcs and miR-1193 promoted DSB repair deficiency and induced apoptosis regulated by the ATR/CHK1/p53 axis (right panel).