All data were expressed in mean ± SD. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate CH5183284 supplier experiments. Statistical significance was determined with Student’s t test (two-tailed) comparison between two groups of data set. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05). Results NAC inhibits NSCLC cell proliferation through reduction of PDK1 protein expression We first examined the effect of NAC on growth of lung carcinoma cells.

A549 NSCLC cells exposed to increased concentrations of NAC for up to 48 h showed a significant decrease in cell proliferation with maximal reduction at 5 mM as determined by Luminescent Cell Viability Assay (Figure 1A). Similar results were observed in other NSCLC cell lines by this (Figure 1B) and as determined by MTT assays BMS-907351 GF120918 molecular weight (Figure 1C). Figure 1 NAC inhibits NSCLC cell proliferation through reduction of PDK1 protein expression. A-B, A549 NSCLC cells exposed to increased concentrations of NAC for up to 48 h (A), or NSCLC cell lines indicated were treated with NAC (5 mM) for up to 48 h (B). Afterwards, cell proliferation was determined by Luminescent Cell Viability Assay. C, NSCLC cell lines indicated were treated with NAC (5 mM) for up to 48 h. Afterwards, cell proliferation was determined by MTT

assays. Data are means ± SD from 3 separate experiments. * p < 0.01, compared with untreated cells (CTR). D-E, Cellular protein was isolated from A549 cells that were cultured with increased concentrations of NAC as indicated for 24 h (D) or cultured with NAC (5 mM) for the indicated time period (E) followed by Western blot analysis with antibodies against PDK1 protein. The bar graphs represent the mean ± SD of PDK1/GAPDH of at least three independent experiments. *indicates significant difference from untreated control (0). F-G, Several NSCLC cells as indicated were treated with NAC (5 mM)

for 24 h followed by Western blot for detecting PDK1 protein. (F) or A549 cells were transfected with control or overexpression of PDK1 vectors for 24 h, followed by exposure of the cells to NAC for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay Kit. The upper panels represent protein levels of Fenbendazole PDK1 by Western blot (G). All data were depicted as mean ± SD. *indicates significant difference as compared to the untreated control cells (CTR). We next determined the effect of NAC on PDK1 protein expression. Cells exposed to NAC resulted in significant decrease in PDK1 protein expression in a dose- and time-dependent manner with maximal induction noted at 5 mM at 24 h as determined by Western Blot (Figure 1D-E). NAC also reduced PDK1 protein expression in other NSCLC cell lines (Figure 1F). Overexpression of PDK1 has been reported to correlate with tumor progression [5].