Figure 5.

Inhibition of CDK4/6 Decreases CHK1 and Induces DNA Damage. (A) Rh30 cells were treated with rapamycin, AZD8055, MK2206 or PD03332991 (1 μM) for 16 hr. Total proteins were extracted for immunoblotting. (B) Rh30 cells were treated with PD03332991 for 12 hr. The total RNA was extracted to detect CHK1 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity of CHK1 mRNA was plotted. Error bars: Mean ± SD (n = 3). **P < 0.01. (C) Rh30 cells were treated with PD03332991 for 16 hr. Total proteins were extracted for immunoblotting to detect γH2AX. (D) Rh30 cells were treated with PD0332991 for 16 hr, then melphalan was added alone or in combination with PD0332991 and incubated for 5 hr. Immunblotting was done to determine CHK1, pCHK1-S345. (E) Rh30 cells were transfected with control (Ctrl) or RB siRNA. 24 hr later, AZD8055 (AZD, 1 μM), HLM006474 (HLM, 10 μM), or AZD8055 plus HLM006474 (AZD HLM) was added for additional 24 hr. Total proteins were extracted for immunoblotting. (F) Rh30 cells were transfected with siRNAs of control, CDK4, or CDK6. 72 hr later, total RNA was extracted to detect CHK1 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity of CHK1 mRNA was plotted. G, Rh30 cells were treated as in Fig. 5F, total proteins were extracted for immunoblotting. GAPDH served as loading controls. Immunoblots were converted to gray with auto tone by Photoshop program.