Fig. 2
From: Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage
DNA damage, DNA damage response (DDR) and apoptosis. A γ-H2AX immunofluorescent staining showed that MEFs expressing viral protein(s) had a higher percentage of γ-H2AX-positive cells than mock cells. However, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) induced higher rates of DNA damage than cells expressing HPV6 E6 alone (6E6). Arrows indicate γ-H2AX positive cells. B The rates of γ-H2AX positive cells were calculated and compared among the clones. Both low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed significantly increased γ-H2AX signals compared with the cells expressing a single viral protein. Asterisk symbol indicates significantly increased rate of γ-H2AX signal (**p < 0.01). C A comet assay showed that viral protein expressing cells induced more comet signals than mock cells. More comet signals and longer comet tails were observed in the cells expressing HPV6 E6 + EBV LMP-1 (6E6 + LMP-1). D Comet tail length of each cell line was measured using ImageJ software. Comet tail lengths of viral protein expressing cell lines were longer than those of mock cells (††p < 0.01), but there was no significant difference between low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). E Low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed less TUNEL signals than mock cells and single low-risk HPV6 E6 (6E6) expressing cells under genotoxic conditions. Arrows indicate TUNEL-positive cells. F TUNEL staining rates of each cell line under genotoxic conditions were measured and compared. Asterisk symbol indicates a significant increase in TUNEL-positive cells as detected by immunofluorescent staining (*p < 0.05). G Under normal conditions, the ATR-ChK1 pathway was induced across all cell lines. However, ATM-ChK2 proteins were not expressed. Under genotoxic conditions, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed suppression in both the ATR-ChK1 and ATM-ChK2 pathways. FBS: fetal bovine serum