Fig. 3

Mechanical overstretch activates PERK signaling in the alveolar epithelium. a. Perfused ex vivo lung complexes ventilated according to NS and OS protocol from Fig. 1 were used. At the end of the experiment lung tissue was incubated with green fluorescent-labeled p-PERK antibody and epithelial cells were labeled with red fluorescent conjugated epithelial- specific occludin antibody. OS significantly increased the number p-PERK positive cells, including those with dual p-PERK and occludin positivity. b The bar graph shows the number of p-PERK positive cells per 100 epithelial cells in NS and OS conditions. c High magnification image of the lung tissue shows p-PERK staining in occludin positive cells (arrows) confirming p-PERK localization in the epithelium. Green = green fluorescent-labeled p-PERK, Red = red fluorescent-labeled occludin, Blue = DPI for nuclear staining. Statistics: Two-way ANOVA was performed with Tukey-Kramer post hoc analysis. N = 4 animals/condition. Using laser scanning confocal microscope N = 5 random images/sample were taken for analysis. We count the number of p-PERK positive cells in 100 occludin positive cells per field and compared them between NS and OS conditions. Images were analyzed by FIJI program. *represents significant increase in the number of dual positive cells, p < 0.05. Data is presented as averaged values ±SEM. Image scale bar = 100 μm (A) and 20 μm (C)