Figure 3. HDACi treatment specifically inhibits CHK1 expression and upregulates its downstream signaling proteins CDC25A, CDC25C, and CDC2, involved in G₂ cell cycle checkpoint control.

A, A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 ^Y15), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B, PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 ^Y15), serine-216 phosphorylation of CDC25C (pCDC25c ^S216), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C, drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D, PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E, A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 ^Y15, and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.