Fig. 6
Blebbistatin inhibits epithelial cell migration in PCLS. Deconvolved widefield, single plane z-stack images from 14 h time-lapse videos (Supplementary movies 14A, B) of control (a–g) and blebbistatin-treated (h, i) P3 PCLS labelled with EpCAM-FITC (green) and SiR-DNA (magenta). Still images are taken from supplementary video 14A at 0 (c), 4 (d), 8 (e) 12 (f) and 14 (g) hours represent the white boxed regions in a and b. Scale bar = 50 µm. MTT cell viability assay comparing control and blebbistatin-treated P3 PCLS at the end of time-lapse, (j), n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment, ns = not significant; paired Student t-test. Individual cell tracking over 14 h in a single field from P3 PCLS treated with control DMSO (k) or blebbistatin (l) containing media. Mean net epithelial cell migration in P3 control and blebbistatin-treated PCLS movies over 14 h (m). n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment. Two fields were quantified per slice. Each dot represents mean net epithelial cell migration per field. Individual net cell migration in blebbistatin treated vs. DMSO control P3 PCLS after 14 h of time-lapse imaging (each dot represents a single cell) (n). A total of 1243 cells for DMSO control and 1340 cells for blebbistatin-treated PCLS were tracked. n = 3 independent experiments using three separate mice, with duplicate slices per condition, per experiment. Two fields were quantified per slice. ***p < 0.0001, **p = 0.001; Mann-Whitney U-test,. Yellow ‘a’ indicates alveolar airspaces. Error bars are defined as s.e.m
