Investigating Epigenetic Regulation in SCLC

The human SCLC cell line NCI-H446 (Cell Bank of Shanghai Institutes for Biological Sciences, China Academy of Sciences, Shanghai, China) was cultured as in a previous study (1 (link)). Once passaged to the fifth generation, the cells were grown in 75 cm² culture flasks and harvested in a solution of trypsin-EDTA at the logarithmic growth phase. A cytometry assay was used for cell counting (2 (link)). For the demethylation treatment, NCI-H446 cells (60–70% confluence) were treated with 5 μm 5-aza-2′-deoxy-cytidine for six days and the medium was replaced every other day. DNA, RNA and protein were extracted for analysis using kits obtained from Sangon Biotech Co., Ltd. (Shanghai, China). For blocking Akt expression, wortmannin (Sigma-Aldrich, St. Louis, MO, USA) was used as an inhibitor of the PI3K/Akt signaling pathway. wortmannin was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and final concentrations of 20 μM were used to treat the cells. Equal volumes of DMSO were used for the vehicle control (17 (link)). CPA-7, which is a specific inhibitor of the PI3K/Akt signaling pathway, was used for blocking STAT3 expression and dissolved in DMSO to achieve a final concentration of 10 μM (18 (link)). For plasmid transfection, pcDNA3-STAT3 and pcDNA3-Akt were received from the Department of Viral-Gene Therapy, Shanghai Eastern Hepatobiliary Surgery Hospital (Shanghai, China).