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. 2021 Feb 22;6(4):e134881. doi: 10.1172/jci.insight.134881

Figure 4. MRCKα is required for the β1 subunit–mediated alveolar barrier tightening.

Figure 4

(A) ATII cells were cotransfected with siRNA (scramble control or against MRCKα) and plasmids (CMV-tet and Tet- β1) 24 hours after isolation. A total of 1 μg/mL doxycycline was added to induce gene expression at day 2. TEER was then measured every 24 hours from day 3 to day 5. ANOVA followed by Bonferroni’s post hoc test was used for statistical analysis, **P < 0.01. (B) ATII cells were cotransfected with plasmids (CMV-tet and Tet-β1) 24 hours after isolation and treated immediately with 2 μM MRCKα inhibitor BDP5290. TEER was measured 24 hours later. Data are presented as mean ± SD. ANOVA followed by Bonferroni’s post hoc test was used for statistical analysis, *P < 0.05. (C) Immunofluorescence staining of zo-1 in cells treated with or without doxycycline for 48 hours after transfection with luciferase plasmid alone, β1 plasmid and siScramble, or β1 plasmid and siMRCKα. Images represent 3 separate experiments. Scale bar: 20 um.(D) Overexpression of MRCKα increases TEER. Data represent n = 6 biological replicates. Data are presented as mean ± SD. ANOVA followed by Bonferroni’s post hoc test was used for statistical analysis, ***P < 0.001.