Figure 4: ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity. | Nature Communications

Figure 4: ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity.

From: Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

Figure 4

(a) Analysis of DiI-LDL uptake in EA.hy926 treated with ACVRL1 siRNA (+) in the presence of increasing amounts of expressed ALK1 (using various MOI of AdALK1-GFP). DiI-LDL uptake was normalized by Hoechst dye stained nuclei. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student’s t-test. (b) ALK1 dose dependently increases DiI-LDL uptake in Ldlr-KO MEFs. DiI-LDL data are normalized for ALK1-GFP expression in Ldlr-KO MEFs by using various MOI of AdALK1-GFP. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student’s t-test. (c) HeLa cells were transfected with either GFP, WT, constitutively active (CA, Q201D) and inactive variant (IA, R374Q) ALK1 constructs and p-SMAD1/5 levels were examined in the absence or presence of BMP9 (10 ng ml−1) (Data are mean±s.e.m., experiment was performed three times). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10a. (d) DiI-LDL uptake analysis of cells expressing GFP and the different variants of ALK1. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student’s t-test. (e) Western bot analysis of p-SMAD1/5 after starvation using BMP9 (10 ng ml−1) or pharmacological inhibitors (ALK1ecto,400 ng ml−1: tenfold molar excess or LDN193189, 50 nM). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10b. (f) BMP9 stimulation or inhibition does not affect DiI-LDL uptake into endothelial cells. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student’s t-test. ns, not significant.

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