Figure 3

OGT activity regulates miR-483-3p expression. (a) MiR-483-3p relative expression by RT–qPCR of HepG2 cells transfected with siRNA for OGT and treated with 2-DG 5 mM for 48 h (lower panel). The lower panel shows the effective OGT protein reduction by siRNA treatment by western blot analysis (b). PrimiR-483 (left y axis) and miR-483-3p (right y axis) relative expression by RT–qPCR, of HepG2 cells treated with azaserine 100 μM for 24 h. (c) Chromatin immunoprecipitation (ChIP) analysis for USF1 occupancy at the promoter of the miR-483 (6841F-7100R) and at the 3′ UTR of UBE3A (3UTR-UBE3A) as control, by RT–qPCR (normalized on input DNA) in OGT-depleted HepG2 cells for 19 h. The BUB1 antibody was used as negative control. (d) (Left) Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract (NE) from HepG2 cells transfected with siRNA for OGT or treated with azaserine 200 μM for 24 h using the miR-483 E-box probe. The specific complexes are indicated with black arrows (b, c). EMSA for lanes siCTRL and siOGT was performed once. (Right) OGT, β-catenin, USF1 protein relative expression and glycosylation status (using RL2 antibody) of the nuclear extract by western blot from HepG2 cells transfected with siRNA for OGT or treated with azaserine 200 μM for 24 h. Densitometry analysis is shown. For statistical analysis, Student’s t-test was applied (**P<0.01; ***P<0.001).