Journal: Nature Communications
Article Title: Angiopoietin-2–integrin α5β1 signaling enhances vascular fatty acid transport and prevents ectopic lipid-induced insulin resistance
Figure Lengend Snippet: Angpt2–ITGα5β1 signaling facilitates FA transport through CD36 and FATP3. a , c , d , g – p HUVECs were treated with vehicle or Mn 2+ (1 mM) + Angpt2 (2.5 μg/ml) for 15 min or indicated time points. a Comparisons of short-chain FA (BODIPY C-5, 8 μM; n = 4) and long-chain FA (BODIPY C-12 and C-16, 8 μM; n = 4 for vehicle and 3 for Mn 2+ + Angpt2) uptake. b Comparisons of depletion efficiency of CD36 , FATP3 , and FATP4 in corresponding siRNA treated HUVECs. n = 3. c , d Comparisons and representative images of FA (BODIPY C-12, 8 μM) uptake in siControl ( n = 7, 5), si CD36 ( n = 4, 5), si FATP3 ( n = 6), or si FATP4 ( n = 6, 8) HUVECs. Scale bars, 30 μm. e Diagram depicting the endothelial FA transport assay. HUVECs were cultured until confluence on trans-wells and FA (BODIPY C-12, 8 μM) was added to upper layer of trans-well, followed by analysis of transported FA in bottom well. f Comparisons of FA transport for indicated time points after Mn 2+ (1 mM) + Angpt2 (2.5 μg/ml) treatment in siControl ( n = 3 for 10, 30 min and 4 for 20 min), si FATP3 ( n = 3) or si FATP4 ( n = 4) HUVECs. n = 3–5. g Comparison of mRNA expression of CD36 and FATP3 . h Representative images of FATP3-td-Tomato transfected HUVECs. Scale bars, 50 μm. i Representative images of CD36 and ITGβ1 expression. Expression of active ITGβ1 (arrows) and CD36 (arrowheads) are co-localized after Angpt2 treatment. Scale bars, 50 μm. j , k Representative images and comparison of in situ proximity ligation assay between CD36 and ITGβ1 complex. Scale bars, 20 μm. l – n Representative images and comparison of in situ proximity ligation assay between CD36 and ITGβ1 complex in HUVECs pre-treated with vehicle or ITGα5β1 blocking peptide (ATN-161, 10 μM) for 15 min. Scale bars, 50 μm ( l ); 20 μm ( m ). o , p Immunoprecipitation with anti-IgG and anti-CD36 antibody in HUVECs. Immunoblot analysis with anti-ITGβ1 and anti-ITGα5 antibodies are shown. Graph indicates normalized ratio of immunoprecipitated ITGβ1 per input. Unless otherwise denoted, each dot indicates a mean of triplicate values from three independent experiments and horizontal bars indicate mean ± SD or SEM ( f ) and P values versus control by two-tailed Student’s t test. NS not significant.
Article Snippet: siRNA transfectionFor siRNA experiments, HUVECs were transfected with siRNAs targeting human CD36 (5′-AAGAGGAACTATATTG-TGCCTCCTGTCTC-3′), FATP3 (Santa Cruz Biotechnology, SASI_Hs01_00100092), FATP4 (Santa Cruz Biotechnology, SASI_Hs01_00047-530), ITGβ1 (5′-TGATAGATCCAATGGCTTA-3′), ITGα5 (Bioneer, 1075709), ITGαV (Bioneer, 1075799), ITGβ3 (Bioneer, 1075875), ITGβ5 (Bioneer, 1075906), TIE2 (5′-GGCUAGUAAGAUCAAUGGUdTdT-3′), NFATc1 (5′-CCCGUUCACGUCAGUUUCUACGUCU-3′)or a scrambled control (5′-UAGCGACUAAACACAUCAA-3′) were used.
Techniques: Transport Assay, Cell Culture, Expressing, Transfection, In Situ, Proximity Ligation Assay, Blocking Assay, Immunoprecipitation, Two Tailed Test