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. 2016 Oct 21;7(47):77764–77776. doi: 10.18632/oncotarget.12794

Figure 5. γH2AX binds to the promoter of the miR-3196 gene and regulates PUMA expression.

Figure 5

A. Schematic diagram of the miR-3196 gene promoter (left panel). The reporter gene plasmid was cotransfected with H2AX or control (vector) into A549 cells and luciferase activity was measured at 48 h after transfection (right panel). pRL-TK Renilla was used as an internal control. Values were normalized to Renilla luciferase activity.** P < 0.01. B. Expression of BIRC7 protein level in H2AX knockdown (upper panel) or overexpression (bottom panel) stable cells after VP-16 (100 μM) induction was evaluated by western blotting. C. BIRC7 mRNA level in H2AX knockdown (right panel) or overexpression (left and middle panels) stable cells treated as in (B) was detected by qRT-PCR. D. ChIPs were performed with IgG (served as a control), anti-H2AX and anti-γH2AX on H2AX-wt or H2AX-139m stable A549 cells treated with VP16 (100 μM) for 48 h. H2AX or γH2AX-associated promoter DNA amounts for miR-3196 were assessed by qRT-PCR with primer pairs flanking the promoter region of the miR-3196 promoter. *** P < 0.001. E. A549 cells were transfected with H2AX alone or combined with miR-3196 mimics (upper panel) for 48 h and PUMA expression was detected by western blotting. A549 cells were transfected with H2AX-siRNA alone or combined with miR-3196-inhibitor (lower panel) for 48 h, and PUMA expression was detected by western blotting. β-actin was detected as a loading control. ** P < 0.01, *** P < 0.001.