Fig. 2: PRMT3 promotes lactate accumulation by methylating PDHK1.
A Lactate levels, triglyceride (TG) levels, total cholesterol (TC) levels, and methionine levels were detected in tumors derived from DEN-CCL4-induced HCC. n = 6 in each group. ** P < 0.01, n.s, no significant difference. B Lactate levels were detected in subcutaneous Hepa1-6 tumors derived from nude mice and C57BL/6 mice. n = 5 or 4 in each group. Each point represents data from one individual mouse. * P < 0.05. C Cell extracts from Huh7 cells with PRMT3 overexpression were subjected to immunoprecipitation using an anti-PRMT3 antibody, followed by immunoblotting to detect PDHK1. D The binding of PRMT3 and PDHK1 in Huh7 cells transfected with HA-PRMT3 and FLAG-PDHK1 were verified by reciprocal immunoprecipitation assays. E The colocalization of PRMT3 and PDHK1 in Huh7 cells was measured by immunofluorescence assay. Scale bar, 20 μm. Pearson’s correlation coefficient (PCC) for evaluating the colocalization of PRMT3 and PDHK1 was analyzed using ImageJ software [72]. The mean PCC R value obtained was 0.82 (R = 0.82). F Representative images of PLA performed in Huh7 cells using antibodies against PDHK1 and PRMT3 were shown. Scale bar, 20 μm. The green dots indicated PDHK1 interacting with PRMT3. G PDHK1 was immunoprecipitated from Huh7 cells treated as indicated, and ADMA levels were subsequently detected by immunoblot analysis. H Representative images of PLA performed in Huh7 cells with PRMT3 overexpression using antibodies against PDHK1 and ADMA were shown. Scale bar, 20 μm. The green dots, as indicated by the white arrows, represent PDHK1 that has undergone ADMA modification. I Wildtype PDHK1, R363K mutant, R368K mutant, R363/ R368K mutant, and their corresponding vector control were transfected into Huh7 cells with PRMT3 overexpression. Subsequently, these PDHK1 variants were immunoprecipitated using Anti-FLAG beads and subjected to immunoblot analysis to detect the level of ADMA. * indicated heavy chain. J The amino acid sequences containing the R363 and R368 sites in various species. K Wildtype PDHK1 and R363/R368K were transfected in Huh7 cells with or without PRMT3 overexpression for 48 hours. Subsequently, PDHK1 and R363/R368K mutations were immunoprecipitated with Anti-FLAG beads and subjected to immunoblot analysis to detect the p-PDHA level. L Wildtype PDHK1, R363/R368K, and their corresponding vector control were transfected in Huh7 cells with PRMT3 overexpression for 48 hours. Then the levels of PDHA, p-PDHA and lactate were detected. M Wildtype PDHK1, R363/R368K, and their corresponding vector control were transfected in Huh7 cells with PRMT3 overexpression for 12 hours. Subsequently, the cells were seeded into a 96-well plate at a density of 5000 cells per well to assess proliferation at the indicated time points. The data are presented as mean ± SD, and the statistical tests were all two-tailed.
