Fig. 2

miR-2355-5p overexpression is HIF-2α dependent. A-B Immunoblot analysis of VHL, HIF-2α, and β-actin and RT-qPCR quantification of miR-210-3p and miR-2355-5p (N = 3) in 786-0, 786-0 VHL, 786-0 VHL Cr.(Crispr)VHL A, and 786-0 VHL Cr.VHL B cells, respectively. C-D Immunoblot analysis of VHL, HIF-1α, HIF-2α, and β-actin and RT-qPCR quantification of miR-210-3p and miR-2355-5p (N = 3) in RCC4, RCC4 VHL, RCC4 shHIF-1α, and RCC4 shHIF-2α cells, respectively. E-F Immunoblot analysis of VHL, HIF-2α, and β-actin and RT-qPCR quantification of miR-2355-5p (N = 4) in 786-0, 786-0 VHL, and 786-0 shHIF-2α cells, respectively. G-H. Immunoblot analysis of VHL, and HIF-2α and RT-qPCR quantification of miR-210-3p and miR-2355-5p (N = 4) in 786-0, 786-0 VHL, and 786-0 Cr.HIF-2α cells, respectively. I-J Immunoblot analysis of VHL, HIF-1α (arrow), HIF-2α, and β-actin and RT-qPCR quantification of miR-2355-5p (N = 3) in 786-0, 786-0 VHL, RCC4, and RCC4 VHL cells cultured for 48 h under normal (21% O₂) or hypoxic (1% O₂) conditions, respectively. K Illustration of the genomic location of miR-2355-5p (GRCh38). L. Expression of KLF7 in ccRCC patient tissues extracted from the TCGA-KIRC (left) and CPTAC3 (right) databases. M Spearman correlation between miR-2355-5p and KLF7 expression in ccRCC patient tissues from the TCGA-KIRC (left) and CPTAC3 (right) cohorts. N Expression of KLF7 in ccRCC patients (CPTAC3) based on tumor VHL status. O RT-qPCR quantification of KLF7 (N = 4) in the cells mentioned in C. P RT-qPCR quantification of KLF7 (N = 3) in the cells mentioned in E. Q RT-qPCR quantification of KLF7 and GLUT1 (N = 3) in the cells mentioned in I. For immunoblot analysis, β-actin and total protein were used as loading controls. The miRNA and gene expression were normalized with RNU44 (B, D,F, H,J) or RPLPO (O, P,Q). The data are presented as the Mean ± SEM. Statistical analysis was performed using two-tailed unpaired Students t-tests (B, D,H, O), one-way ANOVA with Tukey’s multiple comparison test (F, P), two-way ANOVA with Tukey’s multiple comparison test (J) or Dunnett’s multiple comparison test compared to 786-0 VHL in 21% O₂ (Q), Wilcoxon signed-rank test (L, N), and Spearman correlation test (M). (ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001)