Fig. 4

miR-711 targeted Ang-1, FGF14 and Cacna1c. a H/R reduced the expression of Ang-1, FGF14 and Cacna1c. Cells were treated with H/R and subjected to ICC or Western blotting with Abs against Ang-1, FGF14 and Cacna1c. Upper panel: Representative images; Bottom: Semi-quantitative results. b FGF14 and Cacna1c expression in cells treated with H₂O₂, CoCl₂ and AA. Cells were treated for 20 μM AA for 3 h followed by 3 h reperfusion, or with 300 μM H₂O₂, and 500 μM CoCl₂ for 24 h, respectively. Expression of Ang-1, FGF14 and Cacna1c was determined by Western blotting or ICC. c Luciferase reporter assays. H9c2 cells were transfected with firefly and Renilla dual luciferase vectors along with miR-711 mimics or miRNA mimic controls. Dual luciferase vectors contained either the wild type or mutated 3′-UTR of Ang-1, FGF14, or Cacna1c. Firefly/Renilla luciferase activities were measured 48 h after co-transfection. n = 3 for each group. *P < 0.05 miR-711 mimic vs. the miRNA mimic control group transfected with the same dual luciferase. WT, wild type; MUT, mutant; d miR-711 mimic decreased the expression of Ang-1. Cells transfected with miR-711 mimic and exposed to cold H/R. Ang-1 expression was detected by Western blotting. e miR-711 mimic decreased the expression of FGF14 and Cacna1c. Cells were transfected with miR-711 mimic, mimic control, miR-711 inhibitor, or inhibitor control for 48 h and then exposed to cold H/R as described in Fig. 2. Cells were fixed and subjected to ICC staining with Abs against FGF14 or Cacna1c Abs. Green: FGF14; Red: Cacna1c. f Quantitative results of ICC for FGF14 and Cacna1c. Fluorescent intensity of FGF14, Cacna1c and DAPI was measured using the ImageJ program. g Expression of Bax and Caspase 3. Cells were transfected with miR-711 48 h prior to AA treatment. Bax and caspase 3 were detected by Western blotting. β-actin was used as a loading control. n = 3, * P < 0.05