Fig. 5

Mechanical stretch-induced ER Ca²⁺ efflux activates PERK in rat primary alveolar type-I like epithelial cells (RAEC-I). Freshly isolated rat epithelial cells were cultured for 4 days to form RAEC-I monolayers. a Monolayers were stretched for 1,3 and 6 h with 25% surface change (S) or used as unstretched controls (NS). At the end of the experiment fixed monolayers were treated with green fluorescent-labeled p-PERK antibody to detect PERK activation. Mechanical stretch significantly increased p-PERK at 1 h and it remained increased at 3 and 6 h. b Quantified data is shown. c Monolayers were pretreated with 20 μM Ryanodine (Ry) or its vehicle 0.01% DMSO (VC) for 1 h and subjected to biaxial stretch for 6 h with 25% surface change (S). Unstretched monolayers served as controls (NS). PERK activation was assessed with immunofluorescence staining as explained above. Mechanical stretch significantly increased p-PERK, which was significantly reduced by Ry pretreatment. Ry did not significantly change p-PERK in NS monolayers. d Quantified data is shown. e Western immunoblotting was performed to measure the effect of ER Ca²⁺ efflux blockade on eIF2α activation. Ryanodine treatment alone did not affect eIF2α (NS/Ry) compared to control (NS). Mechanical stretch (25%, 6 h) significantly increased p-eIF2α (S). Ryanodine treatment prevented stretch-induced eIF2α phosphorylation (S/Ry). f Quantified data is shown. Statistics: Two-way ANOVA was performed with Tukey-Kramer post hoc analysis for immunofluorescence studies. N = 4 biological monolayer replicates/condition, p < 0.05. Green fluorescent-labeled p-PERK signal was quantified using Fiji by threshold selection and average signal was calculated over 3 different fields of view. Data is presented as average signal ±SEM. Scale bar = 20 μm. For western immunoblot studies, Kruskal-Wallis test was performed for multiple group comparison, and intergroup differences were analyzed with Wilcoxon rank sum test. N = 4 biological monolayer replicates/condition, p < 0.05. Data is presented as averaged values ±SEM. *represents significant increase in green fluorescence S vs. NS conditions. #represents significant decrease between S and S/Ry conditions