Figure 4
PDCD4 is a target gene of miR-21. (a) Western blotting analyzed PDCD4 in H9C2 cells after different concentrations of H2O2 treated (N=4, **P<0.01, ***P<0.001 versus 0 μM). (b) One hundred micromolar H2O2-treated H9C2 cells transfected with miR-21 mimics, inhibitors and both negative controls, western blotting analyzed PDCD4 protein levels in H9C2 cells (N=3, *P<0.05, **P<0.01, ***P<0.001 versus blank group). (c) Schematic representation of 3′-UTR of PDCD4 mRNA reporter with and without the miR-21 seed-binding site (red). (d) Luciferase activity assay of HEK293T cells transfected with luciferase constructs containing WT-3′UTR and Mut-3′UTR of PDCD4 (N=3, ***P<0.001 versus blank group). (e) Western blot analyzed the protein levels of caspase-3 and PDCD4 after H9C2 transfected with siRNA-PDCD4 (N=3, **P<0.01 versus H2O2 group, #P<0.05, ##P<0.01 versus siRNA-PDCD4 group). (f–h) Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining. The proportion of dead cells (Annexin V−/PI+), live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−) and late apoptotic/necrotic cells (Annexin V+/PI+) was respectively measured for comparison. (j)The percentage of apoptotic cells was represent for both early and late apoptotic cells (N=3, **P<0.01 versus siRNA-PDCD4 group)
