Fig. 5

Endothelial LAT1 expression induced by pro-angiogenic factors and its contribution to proliferation and translation regulation. a and b LAT1 mRNA expression in HUVECs starved for VEGF-A and FGF-2 followed by stimulation for 2 h with VEGF-A or FGF-2 alone (10 ng/mL), or with their combination (a), or for 24 h with VEGF-A and FGF-2 (10 ng/mL each) (b). c LAT1 protein expression in HUVECs starved as in (a and b), then stimulated with VEGF-A and FGF-2 (10 ng/mL each). Cell lysates were analyzed by western blotting. β-actin; loading control. Bar graph; densitometric quantification of the band intensity. d LAT1 knockdown (KD) in HUVECs. Crude membrane fractions prepared 48 h after transfection of control (NC) or LAT1-targeting siRNAs (LAT1 siRNA #1–3) were examined by western blotting. Na⁺/K⁺-ATPase α 1; loading control. e and f Cell proliferation of LAT1 KD cells (e) and JPH203-treated cells (f). g and h Effects of LAT1 KD (g) and JPH203 (h) on mTORC1- and GAAC pathways. Cell lysates were prepared and analyzed 48 h after transfection of siRNAs (g), and after JPH203-treatment for 24 h (h). a, b, n = 4; c, n = 3; e, f, n = 8