Fig. 4: RAB5IF exerts significant pro-angiogenic activity in cultured retinal microvascular endothelial cells.

A–E The human retinal microvascular endothelial cells (hRMECs) expressing the applied RAB5IF shRNAs (shRAB5IF#1, shRAB5IF#2 and shRAB5IF#3, with different shRNA sequences against RAB5IF) or the scramble control shRNA (shC) were established, expression of RAB5IF mRNA and protein was shown (n = 5 biological repeats, 15 μg protein/lane for WB) (A); Cells were further cultured for the indicated time periods, sprouting (B), cell proliferation (by testing EdU-positive nuclei ratio, C), migration (Transwell assays, D) and the capillary tube formation (E) were tested by the listed assays. F–J Stable hRMECs expressing the listed lentiviral CRISPR/Cas9-RAB5IF-KO construct (koRAB5IF#1/koRAB5IF#2, with different sgRNA sequences against RAB5IF) or the control construct with non-sense sgRNA (koC) were established, and RAB5IF protein expression was tested (n = 5 biological repeats, 15 μg protein/lane for WB) (F); Cells were further cultured for the indicated time periods, sprouting (G), cell proliferation (by testing EdU-positive nuclei ratio, H), migration (Transwell assays, I) and the capillary tube formation (J) and were tested. K, L Stable hRMECs with the designated genetic modifications on RAB5IF were cultured and subjected to Seahorse analyses to evaluate oxidative phosphorylation (K) and glycolysis (L). M–R Stable hRMECs with the lentiviral RAB5IF-expressing construct (oeRAB5IF-Sl1/oeRAB5IF-Sl2, two stable cell selections) or the empty vector (Vec) were established, expression of RAB5IF mRNA and protein was shown (n = 5 biological repeats, 15 μg protein/lane for WB) (M); Cells were further cultured for the indicated time periods, sprouting (N), cell proliferation (by testing EdU-positive nuclei ratio, O) and migration (Transwell assays, P) were tested, with results quantified. Seahorse assays were also carried out to evaluate ATP contents (Q) and basal glycolysis levels (R). Data are presented as means ± S.D., n = 5 (biological repeats). “N.S.” indicates no statistical difference (P > 0.05); One-way ANOVA with Bonferroni’s post hoc test. Scale bars = 100/200 μm. Source data are provided as a Source Data file.