HU induces hMYH and hRad9 expression. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of hMYH and hRad9 mRNA expression. Total RNA was extracted from untreated or treated (HU or H2O2) cells and used for RT-PCR. The levels of hMYH and hRad9 mRNA were determined and normalized to β-actin levels (unpaired t-test; p < 0.05; *significant; **not significant) (B) hRad9 and hMYH expression in HEK293 cells increased after HU treatment, leading to cell cycle arrest at the G1/S phase. Total cell lysates from untreated, HU-treated, or H2O2-treated cells were used for immunoblotting to measure the expression of hMYH, hRad9, ATR, p-Chk1 (S345), Chk1, p-Cdk2 (T14, Y15), and Cdk2 proteins. (C) hRad9 and hMYH knockdown reduced the phosphorylation of Chk1 and Cdk2 in HU-treated cells. HEK293 cells were transfected with siRNA for GFP, hMYH, hRad9, or hMYH and hRad9 as indicated in the figure. Transfected cells were treated with 20 mM HU for 1 h, and then allowed to recover in fresh medium for 2 h. Total cell lysates were used for immunoblotting analysis with anti-hMYH, anti-hRad9, anti-ATR, anti-p-Chk1 (S345), anti-Chk1, anti-p-Cdk2 (T14, Y15), anti-Cdk2, and anti-actin antibodies.