Figure 3. ALK1 deficiency does not affect sterol sensing in the endothelium.

(a) Quantitative PCR analysis of SREBP2-dependent genes after knockdown of DNM2 or ACVRL1. The loss of DNM2 increases SREBP2-dependent gene expression, whereas the loss of ALK1 does not. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (b) Western blot analysis of the BMP9 (10 ng ml⁻¹) induced phosphorylation of SMAD 1/5. HUVEC were incubated in LPDS and exposed to BMP9 for 60 min. In lane 2, cells were pretreated with LDL (25 μg ml⁻¹) to downregulate LDLR. In lanes 3 and 4, ALK1 or LDLR was silenced with siRNA, respectively. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9b. (c) The loss of ALK1 does not influence LDLR on the cell surface. Flow cytometric analysis of cell surface LDLR levels in endothelial cells treated with control, ACVRL1 or LDLR siRNAs. The Ab C7 was used for LDLR and IgG is an isotype control and data quantified in right panel. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (d) ¹²⁵I-LDL internalization and degradation in cells treated with control, ACVRL1 or LDLR siRNAs. EA.hy926 cells were pre-incubated with LDL (25 μg ml⁻¹) overnight and the internalization and degradation of ¹²⁵I-LDL was after 4 h of incubation. ACVRL1 siRNA reduced internalization and had no effect on LDL degradation, whereas the LDLR siRNA (as a positive control) reduced LDL internalization and led to less degradation of LDL. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (e) Loss of ALK1 does not increase cellular free cholesterol. Filipin-III staining was examined in endothelial cells treated with siRNAs for ACVRL1, LDLR, DNM2 and NPC2 siRNA or treated with U18666 to enhance free cholesterol. Scale bar, 10 μm. Data are representative of at least four experiments. ns, not significant.