Figure 2.

Induction of apoptosis by Rhy in HepG2 cells. (A–C) Rhy modulates expression of various proteins involved in anti-apoptosis, proliferation, angiogenesis, metastasis, and pro-apoptosis. HepG2 cells (1 × 10⁶ cells/well) were incubated with Rhy (130 µM) for 0, 12, 24, 36, and 48 h. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against Bcl-2, Bcl-xL, survivin, inhibitors of apoptosis protein (IAP)-1/2, COX-2, Cyclin D1, VEGF, MMP-9/2, Bax, and p21 as described in the Materials and Methods. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading; (D,E) HepG2 cells (1 × 10⁶ cells/well) were treated with Rhy (130 µM) for 0, 12, 14, 36, and 48 h. Thereafter, equal amounts of lysates were analyzed by Western blotting analysis using antibodies against caspase-8/9/3 and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading; (F) Cells were transiently transfected with pEGFP-Bcl-2 or pEGFP (control vector) plasmid. Bcl-2 protein was overexpressed in pEGFP-Bcl-2 transfected HepG2 cells compared with control. The results shown are representative of the three independent experiments; (G) Transiently transfected cells were treated with Rhy for 48 h. Thereafter, equal amounts of lysate were analyzed by Western blot analysis using antibodies against PARP and β-actin.