Fig. 6 | Scientific Reports

Fig. 6

From: YTHDF1-regulated ALOX5 in retinal pigment epithelial cells under hypoxia enhances VEGF expression and promotes viability, migration, and angiogenesis of vascular endothelial cells

Fig. 6

YTHDF1 depletion downregulates the expression of ALOX5 protein in hypoxia-stimulated ARPE-19 cells. (A) GO enrichment analysis of these pull-down proteins of the mix of Bio-3′UTR. (B) Venn diagram revealing the putative RBPs pulled down by ALOX5 3′UTR. List 1: the 14 proteins associated with mRNA stability in these pull-down proteins; List 2: the putative RBPs of ALOX5 predicted by online program starbase. (C) Representative western blotting images depicting YTHDF1 abundance in the pull-down proteins of Bio-3′UTR and Bio-NC groups. (D) qRT-PCR of YTHDF1 mRNA expression in ARPE-19 cells after transfection by vector of shYTHDF1#1, shYTHDF1#2, shYTHDF1#3, or shNC. (E) Relative ALOX5 mRNA expression by qRT-PCR in shNC- or shYTHDF1-transfected ARPE-19 cells under hypoxia or normoxia, with β-actin as a reference gene. (F) ARPE-19 cells transfected with shYTHDF1 vector or shNC control were exposed to normoxia or hypoxia conditions for 24 h and then treated with Actinomycin D for various time frames, followed by the detection of ALOX5 mRNA level. (G) Relative ALOX5 protein level in shNC- or shYTHDF1-transfected ARPE-19 cells under hypoxia or normoxia. (H) VEGF secretion levels in ARPE-19 cells transfected with shNC- or shYTHDF1 were detected by ELISA under hypoxia or normoxia conditions. Representative pictures were shown. GAPDH served as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001.

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