Figure 5

R2 induced expression of p53 targets. A. Induction of p53 targets in HCT116 and MCF-7 cells. The HCT116 p53⁺/⁺ cells (left panel) and MCF-7 (right panel) were treated with different doses of R2 and Western blotting was performed with p53, Mdm-2, Bax, PARP-1 and caspase-8 antibodies. R2 induced expression of p53 targets in a dose-dependent manner in HCT116 and MCF-7 cells. The affected proteins by R2 are shown by arrows. The densitometry quantitation was performed with Scion Image software. The protein level was measured and expressed relatively for the beta-actin control, and then normalized to untreated sample, which was equal to one. B. Immunostaining demonstrated that R2 activates p21 and increased nuclear localization of p53 and p21 proteins in HCT116 p53⁺/⁺ cells, but not in p53^-/^- cells. Immunostaining with primary p21 (upper panel) or p53 (lower panel) and with secondary Texas-Red conjugated antibodies was performed on HCT116 p53⁺/⁺ and p53^-/^- cells either untreated or treated with R2. The Phalloidin-FITC stained actin was used to observe cell morphology. R2 increased nuclear p53 and p21 in HCT116p53⁺/⁺ cells treated with R2 in contrast to HCTp53^-/^- cells. C. R2 increased G1 arrest in R2-treated cells. Flow Cytometry analysis was performed as described in Materials and Methods on HCT116 p53⁺/⁺ and p53−/− cells that were either untreated or treated with different doses of R2 for 24 h. R2 increased G1-arrested cells and decreased G-2 arrested cells in p53⁺/⁺ cells but not p53^-/^- cells.