Fig. 2
Lactate-induced Snail is pH dependent. a RNA was extracted and subjected to qRT-PCR in A549 and H1299 cells with indicated lactate treatment. Values represent the relative reduction of Snail mRNA normalized to GAPDH. The bars represent the mean ± s.d. of triplicates.**P < 0.01. b A549 and H1299 cells were co-transfected with Snail promoter and control Renilla luciferase reporter gene plasmid and treated with indicated lactate concentrations after 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. **P < 0.01. c Transfection of A549 and H1299 with GPR81 cDNA, GPR81 siRNAs or their respective controls for 48 h, and then were treated with lactate (0 and 20 mM) for 3 h before western blotting. Lactate-induced Snail significantly reduced in cells overexpressing GPR81 cDNA and vice versa. d The cells were pre-treated with MCT1 inhibitor, a-cyano-4-hydroxycin-namate (CHC) or DMSO before with indicated lactate treatment. Western blot demonstrates Snail significantly elevated in CHC-treated group. e The pH of the cell culture medium decreased after lactate treatment. f Snail was markedly induced by pH 5.5 in A549 cells and by pH 6.0 in H1299 cells. g Snail returned to basal levels in the case of adding NaHCO3 into culture medium back to pH 7.35. h A549 and H1299 cells were treated with CHC for 4 h or (i) transfected with GPR81 siRNA for 48 h following culturing cells with fresh serum-free medium for 5 h and then pH and extracellular lactate concentration were measured by pH meter (upper panel) and by Lactate Colorimetric/Fluorometric Assay Kit lower panel), respectively. Treatment of cells with CHC and GPR81 siRNA resulted in a decrease in pH and an increase in lactate concentration. j A549 and H1299 cells were transfected with control and LDHA siRNAs. After 2 days, cells were treated with glucose (0, 2.7 and 4.5 μg/μl) for 3 h before western blot analysis. The levels of Snail induced by glucose was down-regulation by LDHA knockdown. k Western blot analysis of Snail in A549 and H1299 cells treated with glucose (0, 2.7 and 4.5 μg/μl) for 3 h with or without 2-DG (25 mM)