Figure 5.

Involvement of HIF-1α in alveolar epithelial cell phenotypic changes induced by hypoxia. Primary rat AECs were cultured in normoxia (Nx) (21% O₂) or hypoxia (Hx) (1.5% O₂) during 6 days in presence or absence of 10 µM YC-1. (A) Immunostaining of ZO-1 (magenta) and TTF1 (cyan) were performed. n = 4 experiments were performed. Isolated primary rat AECs were cultured in normoxia (Nx) (21% O₂) or hypoxia (Hx) (1.5% O₂) during 6 h in the presence or absence of 100 mM 4-phenylbutyrate (4-PBA) or pre-treated or not with 1 µM BAPTA-AM 90 min before exposition to hypoxia. A representative picture of at least n = 4 independent experiments for each condition has been presented and scale bar represents 50 µm. (B) Western blot of HIF-1α protein levels was performed. Representative blot of n = 5 experiments is shown. Expression levels of HIF-1α were quantified and reported to β-actin expression for each condition. Primary rat AECs were transfected with plasmid coding for luciferase reporter activity of hypoxia responsive element (HRE: i.e., HIF-luc), and cultured as described. (C) Luciferase activity corresponding to the transcriptional capacity of HIF was reported (n = 4 experiments). Raw data were submitted a Kruskal-Wallis test. * and ** indicate a significant difference as compared with normoxic value with p < 0.05 and p < 0.01 respectively. # and ## indicate a significant difference as compared with value in untreated hypoxic cells with p < 0.05 and p < 0.01, respectively.