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. 2016 Nov 21;7:13516. doi: 10.1038/ncomms13516

Figure 2. Validation of ALK1 in mediating LDL uptake into endothelium.

Figure 2

(a) Quantitative PCR analysis for the knockdown efficiency of the ALK1 siRNA in human endothelial cells (HUVEC). Data represent the mean±s.e.m. and are representative of three experiments in duplicate. *P<0.05, Student's t-test. (b) Western blot analysis showing the knockdown efficiency of the ACVRL1 siRNA in HUVEC based on the BMP9 (10 ng ml−1) induced phosphorylation of canonical SMAD 1/5 phosphorylation. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9a. (c) DiI-LDL uptake was reduced in various human (EA.hy926 and HUVEC) and mouse (MLEC) endothelial cells treated with ACVRL1/Acvrl1 siRNA. Scale bar, 50 μm. (d) 125I-LDL uptake into WT and Acvrl1fl/fl/Ldlr−/− MLEC. Acvrl1fl/fl/Ldlr−/− MLEC were infected with AdGFP (control) or AdCre/GFP, then the uptake (includes bound LDL) of 125I-LDL was examined. Cells were kept in LPDS overnight before uptake studies. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (e) Cells were treated with control siRNA, LDLR siRNA or ACVRL1 siRNA and placed into regular media, washed and exposed to increasing concentrations of DiI-LDL. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (f) Uptake analysis of DiI-HDL, -LDL and -VLDL (2.5 μg ml−1) into endothelial cells treated with control siRNA and ACVRL1 siRNA. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (g) Uptake of oxidized LDL (2.5 μg ml−1) into EA.hy926 cells treated with control siRNA and ACVRL1 siRNA cultured overnight in LPDS media supplemented with 25 μg ml−1 LDL. Data represent the mean±s.e.m. and are representative of three experiments. (h) Comparison of DiI-LDL uptake into Ldlr-KO MEFs transfected with either GFP (negative control), ALK1-GFP, ALK2-GFP or LDLR-GFP (positive control). Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (i) Uptake of DiI-LDL in EA.hy926 cells in the presence of increasing concentrations of ALK1-Fc was measured. Addition of 10−9 M ALK1-Fc is equimolar to DiI-LDL (500 ng ml−1). Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test.