Fig. 1: FZD4 is significantly upregulated by SMAD4 in porcine GCs.
From: SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis
a FZD4 mRNA expression level (fragments per kb of exon per million fragments mapped; FPKM) in porcine GCs under treatment with siRNA (control) or siSMAD4 (SMAD4-KD) according to RNA-seq. b FZD4 mRNA expression in porcine GCs under treatment with SMAD4-siRNA (siSMAD4) or SMAD4-overexpressing vector (SMAD4OE) was assessed by qRT-PCR. c FZD4 and β-catenin protein levels in porcine GCs after SMAD4 overexpressed or knockdown were measured by western blot (left), quantification was shown in right panel. GAPDH was used as a loading control. d The correlation between SMAD4 and FZD4 mRNA level was detected in 15 porcine ovarian follicles. The ΔCt values were subjected to Pearson correlation analysis. e qRT-PCR validation of FZD4 mRNA levels in porcine GCs after specific FZD4 knockdown with two small-interfering RNAs against siFZD4-1 and siFZD4-2. f Representative western blot analysis (left) and quantification (right) of FZD4, β-catenin and cleaved Caspase-3 (c-Caspase-3) protein levels in FZD4-silenced porcine GCs. GAPDH was used as a loading control. g FZD4 protein levels in porcine GCs co-transfected with siFZD4 and SMAD4OE were analyzed by western blot. Images (top) and quantification (bottom). h Representative FACS analysis of the apoptosis rate of porcine GCs after FZD4 knockdown or with SMAD4 ectopic expression. i Representative western blot images (top) and analysis (bottom) for c-Caspase-3 expression in porcine GCs treated with siFZD4 or siFZD4 with SMAD4 overexpression. Con. indicates control. Throughout, results are shown as mean ± S.E.M. (n = 3, each). **p < 0.01, ns indicates no significance versus scrambled or control.
