Figure 4

Cyclopamine treatment promotes G1 arrest. A, HOS and143B cells were treated with 10 μM cyclopamine. After 48-hour treatment cells were collected and subjected to cell cycle analysis. When 143B cells were cultured without cyclopamine, 39.8% of cells were in G1 phase. On the other hand, when cultured with cyclopamine, 56.6% of cells were in G1 phase. In the case of HOS cells cultured without GSI, 55.4% of cells were in G1 phase, while 72.3% of cells were in G1 phase when treated with cyclopamine (error bar means S.D.). B, Real-time PCR was performed to quantify mRNAs of cell cycle related genes. Twenty-four-hour treatment with cyclopamine reduced levels of cyclin D1, Cyclin E1, SKP2, and NMYC transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. C, Western blot analysis of levels of cell cycle-related genes. Forty-eight-hour treatment with cyclopamine reduced levels of expression of cyclin D1, cyclin E1, SKP2, and phosphprylated RB (pRb) proteins. Expression of P21^cip1 protein was upregulated by cyclopamine treatment. The experiment was triplicate with similar results (cyclopamine: 10 μM).