Figure S5. Absence of Egfr in murine lung ECs increases the number of pro-inflammatory macrophages.

(A, B) Lung sections of mice conditionally lacking Egfr in ECs were stained for CD68 (green), iNOS (white, A) and Arg1 (red, B). (A, B) The numbers of iNOS⁺ (A) and Arg1⁺ (B) macrophages were quantified by confocal imaging. (C, D, E) Human pulmonary arterial endothelial cells (PAECs) were transfected with either scrambled siRNA (siCTL) and siRNA against AREG (siAREG) and placed in a transwell chamber. They were then cultured in hypoxic conditions with or without leukocytes for 24 h. (C) AREG expression was assessed in leukocytes and HPAECs by qPCR. (D) Apoptosis of normoxic and hypoxic HPAECs was quantified by flow cytometry and shown as fold change compared with the level of apoptosis in siCTL PAECs. (E) HPAECs were plated on Matrigel, and tube formation was measured. (F) Human PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. PAECs apoptosis was assessed by measuring caspase 3⁺ cells and caspase 3 MFI by flow cytometry. Isotype control was used to determine caspase 3 positivity. n = 5 replicates per condition. Data are shown as mean. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.