Extended Data Fig. 9: Progressively increased doses of rock inhibitors causes progressingly increased cytoskeletal actin disassembly in LF primary cells; at low concentrations, ripasudil rock inhibition uncouples the LF cell’s sensing and alignment in the direction of experienced stretch and results in fewer SMAα cells in conditions with and without 2-dimensional stretch.
A, LF primary cells treated with increasing doses of rock inhibitors stained for actin (top) or SMAα (bottom). Insets contain schematics emphasizing morphology observed with each level of treatment: full-length fibers in untreated cells (i,iv) dissolve into fragments (ii), then into sparse granules on the periphery of the cell cytoplasm (iii,v). B, LF primary cells were assigned conditions of ‘no stretch’ or ‘stretch’ and then treated with vehicle control or ripasudil. After stretch regimen, cells were stained for actin and SMAα (i-iv; insets contain representative schematics of the actin fibers directions of cells) and the percentage of SMAα cells was quantified (v). Percent SMAα cells increased with stretch, and decreased with ripasudil treatment, in both stretched and unstretched conditions. C, Immunofluorescence staining for SMAα and DAPI in paired, unstretched and stretched LF fascicles, with quantification of n = 12 triply paired fascicles. Images and graph in c have been reproduced from main manuscript Figs. 4g and 5f, respectively, for efficient comparison to results in b,i-v. Quantitative data are shown as mean ± sem.
