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. 2013 Dec 21;5(9):2404–2417. doi: 10.18632/oncotarget.1650

Figure 7. Ad-FIRΔexon2, rather than Ad-FIR, increased BLM-induced DNA damage as indicated by γH2AX in HLE and HLF cells.

Figure 7

(A) After 48h of bleomycin (BLM), Ad-FIR (FIR), Ad-FIRΔexon2 (Δ2) single treatment or co-treatment, whole-cell lysates of HLE cells were extracted and analyzed by Western blotting. For the co-treatment, BLM and Ad-FIR or Ad-FIRΔexon2 were co-treated at starting point and incubated for 48h (lanes 4 and 5). Ad-FIR (FIR) or Ad-FIRΔexon2 (Δ2) was added after 24h of BLM treatment, then incubated for another 24 h (lanes 8 and 9). After 24 h of Ad-FIR or Ad-FIRΔexon2 treatment, BLM was added, then incubated for another 24 h (lanes 10 and 11). BLM was added at a concentration of 30 μg/ml. Ad-GFP, Ad-FIR and Ad-FIRΔexon2 were added at a concentration of 3.76 × 108 VP/ml (10 MOI). Lane 1, mock (untreated cell lysate); lane 2, Ad-GFP (GFP) as negative control; lane 3, BLM; lanes 4, 8 and 10, BLM and Ad-FIR; lanes 5, 9 and 11, BLM and Ad-FIRΔexon2. (B) HLE cells were treated with BLM (30 μg/ml) and/or Ad-GFP (3.76 × 108 VP/ml; 10 MOI), Ad-FIR (3.76 × 108 VP/ml; 10 MOI) or Ad-FIRΔexon2 (7.52 × 108 VP/ml; 20 MOI) for 48 h, and whole-cell lysates were extracted and subjected to Western blotting. Lane 1, Ad-GFP (GFP); lane 2, BLM; lane 3, BLM with Ad-FIR; lane 4, BLM with Ad-FIRΔexon2; lane 5, Ad-FIR alone; lane 6, Ad-FIRΔexon2 alone. (C) P27Kip1 and γH2AX expression was examined in HLE and HLF cells by immunocytochemistry staining following BLM (100 μg/ml) treatment alone or with Ad-FIRΔexon2 (7.52 × 108 VP/ml; 20 MOI).