<i>A</i>, HDACi-mediated decrease in CHK1 precedes apoptosis.

<p>A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 ^Y15), Ser216 phosphorylation of CDC25C (pCDC25 ^S216), acetyl-H4, and cyclin B1 were determined. <i>B</i>, Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. <i>C</i>, Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 ^Y15, and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. <i>D</i>, in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP <i>ex vivo</i>. Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.