Figure 4. Gain-of-function experiments for tylophorine-induced decreased protein expression of cyclin D1, cyclin D2, and c-Myc.

A. Ectopically overexpressed cyclin D1 or D2 restored the hyperphosphorylation of pRb and the expression of cyclin A2. B. Ectopically overexpressed c-Myc rescued the biological function of its downstream effectors (e.g., cyclin B1 and p-pRb) but not the protein expression of cyclin D1 and D2. C. Ectopically overexpressed cyclin A2 moderately restored the hyperphosphorylation of pRb. HONE-1 cells were transfected with the indicated expression vectors for 24 h prior to treatment with either vehicle (DMSO) or tylophorine at the indicated concentrations for an additional 24 h. The resulting cell lysates were analyzed by western blotting with the indicated antibodies. The relative expression or phosphorylation levels of each protein were normalized with their respective internal loading control α-tubulin or GAPDH. The results shown are representative of 3 independent experiments.