Fig 9. FEN1 plays a vital role in DNA damage and cell death induced by knockdown of hWH.
(A) Depletion of FEN1 mitigates DNA damage and cell death resulting from hWH knockdown in human cells. Double knockdowns of hWH and FEN1 were conducted in HCT116 p53+/+ cells by siRNAs. We utilized the siRNA pool to knockdown hWH, and three different single siRNAs for FEN1 knockdown. The depletion efficiency of hWH and FEN1, and the DNA damage level revealed by γ-H2AX were determined by western blot. Viability of each treatment was measured by MTT assay. Two of the FEN1 siRNA showing better FEN1 knockdown efficiency (sihFEN1-1 and -2, lanes 2 and 3) were able to better rescue DNA damage and cell death induced by hWH knockdown (lanes 6 and 7). (B) Ectopic expression of FEN1 reverses the rescuing effect by FEN1 knockdown and makes hWH knockdown more toxic. Transfection of vector for ectopic expression of FEN1 tagged with myc and DDK was conducted with pCMV6/FEN1-myc-DDK construct (0.2 μg/ml for 105 cells). Knockdowns of hWH and FEN1 were carried out with siRNA pools. While knockdown of FEN1 can rescue cytotoxicity induced by hWH knockdown (lanes 4 versus 6), ectopic expression of FEN1 can restore the cytotoxicity of hWH knockdown (lanes 6 versus 8). Interestingly, combined expression of FEN1 from both endogenous and ectopic sources exacerbates cytotoxic effect of hWH depletion (lanes 4 versus 7).