Fig. 5.

Osmotic stress in cystic fibrosis (CF) small airway epithelial cells (SAECs) results in enlarged nuclear structures and affects high-mobility group box-1 protein (HMGB-1) distribution. A and B, top: subcellular localization of HMGB-1 labeled with primary monoclonal rabbit anti-HMGB-1 and corresponding secondary goat anti-rabbit IgG conjugated with Alexa Fluor 488 (green) in normal and CF SAECs under isosmotic conditions. A and B, bottom: conjugated GFP secondary antibody signal was changed to binary (black and white) images to facilitate and contrast view of HMGB-1 subcellular location under isosmotic and hyposmotic solutions. C: hypotonic (HYPO) saline significantly increases nuclear size in normal SAECs (n = 47) vs. isotonic (ISO) control (n = 70). *P < 0.01. Nuclear size of CF SAECs did not change significantly in isotonic (n = 82) vs. hypotonic (n = 37) solution. Cell observations from 3 independent studies analyzing 4 regions of interest are reported. Statistical comparisons vs. normal SAECs in isotonic saline were conducted by ANOVA followed by post hoc testing (Holm’s t-test).