Figure 3
Tensile forces drive collective migration by activating mechanically sensitive intracellular signaling and transcription factors.
(a) Phase contrast images showing collective migration from representative tissues treated with DMSO (control) or blebbistatin (12.5 μM). (b) Quantification of cohort length from tissues treated with DMSO or blebbistatin. Mean ± s.e.m. of three replicates is shown, n = 60 tissues per group. **P < 0.01, Student’s t-test. Immunofluorescence staining for FAK pY397 and phospho-p130Cas in representative DMSO (c) and blebbistatin-treated (d) migrating cohorts. (e) Confocal image showing immunofluorescence staining for MRTF-A localization (green) and nuclei (red) in a representative tissue. (f) Quantification of the localization of MRTF-A (nucleus/cytoplasm) in tissues. Levels of nuclear and cytoplasmic MRTF-A were quantified by measuring signal intensity in the two compartments. Mean ± s.e.m. of three replicates is shown. Migrating cohort (invasion): n = 14; quiescent body: n = 11. **P < 0.01, Student’s t-test. (g) Confocal image showing immunofluorescence staining for MRTF-A localization (green) and nuclei (red) in representative tissues treated with DMSO or blebbistatin. (h) Quantification of the localization of MRTF-A (nucleus/cytoplasm) in tissues treated with DMSO or blebbistatin. Levels of nuclear and cytoplasmic MRTF-A were quantified by measuring signal intensity in the two compartments. Mean ± s.e.m. of three replicates is shown. MRTF-A DMSO: n = 29; MRTF-A blebbistatin: n = 21; DAPI DMSO: n = 10; DAPI blebbistatin: n = 12. **P < 0.01, Student’s t-test. (i) Phase contrast images showing collective migration from representative tissues treated with DMSO or CCG-1423 (10 μM). (j) Quantification of cohort length from tissues treated with DMSO or CCG-1423. Mean ± sem. of three replicates is shown. Invading cohorts treated with DMSO: n=80; invading cohorts treated with CCG-1423: n = 57. **P < 0.01, Student’s t-test. (k) Frequency maps showing collective invasion from 34 tissues (three independent replicates) transfected with scrambled shRNA (shScr) or shMRTF-A (two constructs). (l) Quantification of cohort length from tissues transfected with shScr or shMRTF-A. Mean ± s.e.m. of three replicates is shown, n = 50 tissues per group. ***P < 0.001, Student’s t-test. All images are representative of three independent replicates in which >50 tissues were monitored. Scale bars, 50 μm (a,i,k), 25 μm (c,d,e,g).
