Fig. 2: Role of XPA-mediated NER in DNA damage response and cytotoxicity triggered by OH-ME.
Transient knockdown of XPA in HepG2. Cells were transfected with XPA siRNA or scrambled (scr) RNA followed by OH-ME treatment. Representative western blot experiments in HepG2 cells with XPA knockdown and subsequent OH-ME treatment (0-75 µM) for 24 h. The samples were analyzed by SDS-PAGE and western blot detection of XPA, γH2AX, pCHK2 (A), p53 and cleaved caspase-3 (B). Hsp90 served as loading control. C Cell death induction after XPA knockdown and OH-ME treatment (0-75 µM) for 72 h in HepG2 cells. Apoptotic and necrotic cell death was determined by Annexin V-FITC/PI staining and flow cytometry (n = 3, except for 50 µM, n = 2). D Impact of siRNA-mediated XPA knockdown and OH-ME treatment on viability in HepG2 cells. Cells were exposed to increasing concentrations of OH-ME (0-75 µM) for 72 h. Viability was then measured by MTS assay (n = 4). E OH-ME triggered DNA damage response in primary murine hepatocytes (PMH) proficient or deficient for XPA. PMH were exposed to increasing concentrations of OH-ME (0–75 µM) for 24 h and subjected to western blot analysis of γH2AX. Hsp90 served as a loading control. A representative western blot is shown (n = 3). F Viability of WT and XPA–/– PMHs after OH-ME treatment (0–75 µM) for 24 h. Cell viability was determined by the resazurin reduction assay (n = 6 for WT, n = 3 for XPA–/–). All data given as mean + SEM (ns p > 0.05, **p < 0.01, ***p < 0.001).
