Augmentation of Gefitinib-Induced Cytotoxicity by miR-634
(A) Caspase-3/7 activity assay. After 24 h of gefitinib treatment, caspase-3/7 activity was measured and normalized to the cell survival rate in each combined treatment group, and the results are reported as the relative rate compared with non-transfected and non-treated cells. (B) Cell survival assay. Cells were treated as described in (A). The results are reported as the relative rate compared with non-transfected and non-treated cells. (C) Combination index for A431 cells treated with gefitinib and miR-634. (D) Western blotting analysis of ASCT2 and apoptosis markers. Cell lysates were separated by SDS-PAGE and immunoreacted with the indicated antibodies. (E–I) Metabolite analysis of A431 cells treated with gefitinib (Gef) and/or miR-634. A431 cells were transfected with 10 nM miR-NC or miR-634 and simultaneously treated with gefitinib (10 μM) for 24 h. Lactate production (E), intracellular glutamine levels (F), intracellular ATP levels (G), the GSH/GSSG ratio (H), and intracellular ROS levels (I) were measured and normalized to the cell survival rate. Error bars indicate the SD. Data are presented as mean ± SD. p values were calculated using two-way ANOVA.