Fig. 6: OGDHL controlled FASN by TFAP2A transcriptional regulation.

The results are expressed as the mean ± SEM of three independent experiments, and there are at least three replicates in each independent experiment. t-test, ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 (independent-samples t-test for statistics). A FASN protein and mRNA levels after TFAP2A overexpression were detected by western blotting and qRT-PCR. B Primers were constructed segmentally according to the predicted binding site of TFAP2A on the FASN promoter region. Agarose gel electrophoresis and qRT-PCR were used to visualize the results of ChIP experiments. C Fluorescence detection of A498 cells transfected with truncated plasmids showed that TFAP2A binds to FASN promoter NO. 2 (−1382 ~ −855) or NO. 3 (−399 ~ −391). D Western blot analysis was used to detect the protein levels of FASN and TFAP2A in OGDHL-overexpressing ccRCC cell lines. E, F Protein stability assay in A498 cells overexpressing OGDHL. Cells were treated with 50 μM cycloheximide (CHX), harvested at specific time points (0 h, 4 h, 8 h, 12 h, 24 h), and the protein levels of OGDHL and TFAP2A were detected by western blot analysis. G A498 and CAKI cells were treated independently with 20 µM MG132 and chloroquine for 12 h, and the expression of TFAP2A protein was analyzed by western blot analysis. H Negative controls and OGDHL lentivirus-infected ccRCC cell lines were immunoprecipitated with TFAP2A antibody and incubated with ubiquitin (Ub) antibody for western blot analysis.