Secretion of VEGFA by immortalized BCi-NS1.1 cells. A. VEGFA levels assessed by ELISA in growth media from primary airway basal cell cultures (n = 4) and immortalized BCi-NS1.1 cell cultures (n = 4). Secreted VEGFA was normalized to cell number and calculated as pg/cell/ml. Data shown is the average ± the standard error of n = 4 independent cultures of primary BC and n = 4 independent passages of BC-NS1.1 cells from passage 10 to 14. Statistics were calculated by a 2-tailed Student’s t test. B. Endothelial cells support the growth of primary airway basal cells and immortalized BCi-NS1.1 cells in the absence of growth factors. Human primary BC and immortalized BCi-NS1.1 cells were co-cultured with Akt-activated human umbilical vein endothelial cells (HUVEC-Akt) in cytokine- and serum-free conditions. At the desired time points, cells were harvested and the GFP-labeled HUVEC-Akt cells was determined as the GFP+VE-cadherin+ population by flow cytometric analysis, and the GFP-VE-cadherin- population quantified as expanded basal cells. Data shown is the average ± the standard error of n = 3 independent cultures of primary BC and n = 3 independent passages of BC-NS1.1 cells from passage 5 to 26 compared directly in tandem for each independent experiment. Statistics were calculated by a 2-tailed Student’s t test.