Fig. 7
Blebbistatin inhibits ex vivo alveologenesis. H&E stained sections from P3 PCLS at 0 and 72 h, cultured with DMSO control (top panels) or 50 μM blebbistatin (bottom panels) (a). Scale bar = 100 µm. Mean linear intercept (Lm) (b) and airspace count (c) obtained from H&E sections of P3 PCLS treated with DMSO control or 50 μM blebbistatin at 0 and 72 h, n = 3 independent experiments using 3 separate mice, 3 H&E sections from each PCLS from each mouse were quantified per group, per experiment, each dot represents per field count (b, c); ***p < 0.001, ns = not significant, one-way ANOVA with Tukey’s post hoc test. Confocal single plane z-stack images of DMSO control (top panels) and 50 µM blebbistatin (bottom panels) P3 PCLS at 0 and 72 h culture, immunostained with Ki67 (red), Sp-C (green) and DAPI (blue) (d). Scale bar = 50 µm. Quantification of Ki67 and Sp-C +ve cells in control and blebbistatin-treated P3 PCLS at 0 and 72 h culture (e), n = 3 independent experiments using 3 separate mice, with duplicate slices per group per experiment. Two fields were quantified per slice. Each dot represents mean value of per field counts per experiment; **p < 0.01; ***p < 0,001; ns = not significant; one-way ANOVA with Tukey’s post hoc test. Quantification of Ki67 +ve cells in DMSO control and blebbistatin-treated P3 PCLS at 0 and 24 h in culture (f), n = 3 independent experiments using 3 separate mice, with duplicate slices per group, per experiment. Two fields were quantified per slice. Each dot represents mean value of per field counts, per experiment; one-way ANOVA with Tukey’s post hoc test. Confocal single plane z-stack images of DMSO control (top panels) and 50 µM blebbistatin-treated (bottom panels) P3 PCLS at 0 and 24 h culture, immunostained with Ki67 (red) and DAPI (blue) (g). Scale bar = 50 µm. Error bars are defined as s.e.m
