Figure 4.

Lactate inhibits glycogen synthase kinase 3 beta/beta‐transducin repeat‐containing proteins (GSK‐3β/β‐TrCP)‐mediated ubiquitination and degradation leading to DNA methyltransferase 1 (DNMT1) upregulation. A, Cells were treated with cycloheximide (CHX) for the indicated time in the presence or absence of lactate. Western blot was used to determine DNMT1 protein levels. B, After cells were transfected with β‐TrCP cDNA, western blot showed DNMT1 protein decreased in a dose‐dependent way. C, Cells were treated with lactate after which cell lysates were immunoprecipitated with anti‐DNMT1 antibody and then western blotted with anti‐ubiquitin. Normal IgG was used as a negative control. D, Expression levels of p‐AKT and p‐GSK‐3β were analyzed by western blotting. E and F, Western blotting shows the levels of DNMT1, transcriptional co‐activator with PDZ binding domain (TAZ) and LATS2 after overexpression with GSK‐3β cDNA (E) and treatment with GSK‐3β inhibitor (F). G and H, Western blotting shows the levels of DNMT1, TAZ and LATS2 after overexpression with AKT cDNA (G) and treatment with LY294002 (H). I, Cells were treated with LY294002, after which cell lysates were immunoprecipitated with anti‐DNMT1 antibody and then western blotted. Normal IgG was used as a negative control. J, Levels of phosphorylation of AKT and GSK‐3β induced by lactate were markedly decreased in the presence of CHC. (Western blotting quantitative analysis of Figure 4 is shown in Figure S4.)