Fig. 4

E2F3 is downregulated by miR-200a in RCC. a Schematic diagram showing the design of constructs containing wild-type and mutant 3’UTR sequence of E2F3 that binding with miR-200a. The miR-200a complementary seed region and the compensatory mutation sites are indicated in red and blue, respectively. b, c Expression of E2F3 in renal cell carcinoma tissues were assessed by western blot (b), and the intensities of individual bands were analyzed by Image J software and normalized with GAPDH to calculate the relative levels of E2F3 (c). d Expression of E2F3 in renal carcinoma cells were detected by western blot. The intensities of individual bands were analyzed by Image J software and normalized with GAPDH to calculate the relative levels of E2F3, as shown at the right bottom. e E2F3 WT-3’UTR or E2F3 Mut-3’UTR pMiR-Report luciferase vector along with the indicated miR-NC, miR-200a mimics, miR-NC inhibitor or miR-200a inhibitor were co-transfected into ACHN cells for 48 h and luciferase assays were performed. Luciferase activity was assessed by normalization of firefly luciferase activity to β-galactosidase activity. f E2F3 protein levels in miR-NC, miR-200a mimics, miR-NC inhibitor, or miR-200a inhibitor transfected ACHN cells were measured by western blot analysis. The intensities of individual bands were analyzed by Image J software and normalized with GAPDH to calculate the relative levels of E2F3, as shown at the right bottom. Results were collected from three independent experiments, with triplicate repeats for each experiment. Data are shown as the mean ± s.d. ^**P < 0.01; ^***P < 0.001