Figure 4.

Calcium chelation prevents the induction of UPR pathways in primary rat alveolar epithelial cells exposed to hypoxia. Primary rat AECs were treated or not with 1 µM 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA-AM) 90 min before a 6-h exposition to normoxia (Nx) (21% O₂) or hypoxia (Hx) (1.5% O₂). (A) Western blot of ATF4 and (B) ATF6N/ATF6 ratio were performed. Representative blot of n = 5 experiments is shown. Quantification of ATF4 and ATF6N protein expression was performed and was reported to the β-actin expression for each condition. (C) Primary rat AECs were transfected with plasmid coding for luciferase reporter activity of amino acid response element (AARE: i.e., ATF4-luc) or (D) endoplasmic reticulum stress element (ERSE: i.e., ATF6N/sXBP1-luc), and cultured as describe. (C) Luciferase activity corresponding to the transcriptional capacity of ATF4 or (D) ATF6N/sXBP1 was measured (n = 4 experiments). Raw data were submitted to a Kruskal-Wallis test. * indicates a significant difference against control or normoxic condition (p < 0.05). # indicates a significant difference as compared with value in untreated hypoxic cells (p < 0.05).