Fig. 3

Celastrol increased p27 protein level in gastric cancer cells through miR-21. Western blot showed that celastrol significantly increased p27 protein level in BGC-823and MGC-803 cells (a). The real-time PCR revealed that celastrol had no effect on p27 mRNA level in BGC-823and MGC-803 cells (b). BGC-823and MGC-803 cells were treated without (control) or with celastrol for 24 h, at which time cycloheximide (50 μg/ml) was added for the indicated periods of time. All the treated cells were then harvested and lysed for western blot analyses to determine the protein levels of p27 and β-actin (as a loading control). Representative immunoblots and a graph showing protein levels of p27 relative to β-actin are shown (c). Western blot analyses revealed that miR-21 mimic reversed the increase of p27 protein level in BGC-823 and MGC-803 cells treated with celastrol (d). **P < 0.01, indicate significant differences from the respective control groups