Figure 4.

Coroglaucigenin induces senescence through the downregulation of CDK4. (A) Immunoblot analysis of CDK4 in HT‐29 and SW480 cells treated with 4 μM of CGN for 24 hours. ImageJ densitometric analysis of the CDK4/β‐actin ratios from immunoblots. (B and C) HT‐29 cells were transfected with siCDK4 or treated with 4 μM of CGN, and then stained for SA‐β‐gal activity 2 d after treatment. (D) CDK4 mRNA expression in cells treated with the indicated concentrations of CGN for 24 hours was evaluated by RT‐PCR. (E) Immunoblot analysis of CDK4 in cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132. ImageJ densitometric analysis of the CDK4/β actin ratios from immunoblots. (F) Confocal microscopy of HT‐29 cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132, then immunostained for Hsp90 (red) and CDK4 (green). Arrows indicate colocalization of green (CDK4) and red (Hsp90) signals. Scale bars, 10 μm. (G) Hsp90 interaction with CDK4 was determined by coimmunoprecipitation assays in HT‐29 cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132. *P < .05, **P < .01