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. 2021 Jul 14;8:680182. doi: 10.3389/fvets.2021.680182

Figure 6.

Figure 6

Experimental validated of ceRNAs regulation with miR-709. (A) Relative expression levels of miR-709, Hif1a, and lnc4040 in GCs between HFs and AFs. (B) Expression of HIF1A protein in GCs between HFs vs. AFs and quantified by ImageJ (v1.45), error bars represent SEM (n = 3) **p < 0.01. (C) Predicted miR-709-binding site on Hif1a and lnc4040, and design of luciferase reporter. (D) 293T cells were co-transfected with wild-type (WT) or mutant (MUT) luciferase reporters of Hif1a with miR-709 mimics, mimics NC, or miRNA inhibitors. The relative levels of firefly luminescence normalized to Renilla luminescence are plotted. Error bars represent SEM (n = 3) *p < 0.05. (E) 293T cells were co-transfected with wild-type (WT) or mutant (MUT) luciferase reporters of lnc4040 with miR-709 mimics, mimics NC, or miRNA inhibitors. The relative levels of firefly luminescence normalized to Renilla luminescence are plotted. Error bars represent SEM (n = 3) *p < 0.05. (F) Expression of HIF1A protein in GCs infected with miR-709 mimics, mimics NC, miR-709 inhibitor, and inhibitor NC and quantified by ImageJ (v1.45); error bars represent SEM (n = 3) **p < 0.01. (G) Expression of HIF1A protein in GCs infected with miRNA mimics, mimics NC, and lnc4040 overexpression vector and quantified by ImageJ (v1.45); error bars represent SEM (n = 3) **p < 0.01.