-ATPase α3 subunit knockdown sensitized T98G cells to TMZ. To test the hypothesis that the high expression of Na+/K+-ATPase, especially its α3 subunit, plays a critical role in drug resistance of glioblastoma cells, we targeted the α3 subunit using the RNAi technique to downregulate its expression in T98G cells. A. RT-PCR assay verified that the α3 subunit was knocked down by the α3 stealth RNAi in T98G cells. B. Trypan blue cell death assay showed that, compared to control (blank bars), α3 knockdown (filled bars) in the drug-resistant T98G cells induced a trend of increased cell death even without drug insult. The addition of TMZ (100 μM) for 48 hrs more than doubled cell death in α3 knockdown T98g cells compared to the TMZ-induced cell death without α3 knockdown. N = 3 for each group. * P < 0.05 vs. controls; #
P < 0.05 vs. TMZ treatment in control cells. C. Western blots of AIF, cytochrome c, β-actin and VDAC in cytosolic and mitochondrial fractions of transfection vehicle control (lipofactamine) and α3 knockdown cells with or without TMZ. There was no noticeable difference in AIF expression under different conditions while TMZ appeared to affect cytochrome c levels in the cytosol and mitochondrial compartments. D. Quantitative analysis of the Western blotting showed that α3 knockdown drastically increased cytochrome c release into the cytosol, while the mitochondrial cytochrome c level decreased in TMZ-treated α3-knockdown cells. N = 3 independent assays in each group. * P < 0.05 vs. α3 knockdown controls.