FIGURE 11.

Myosin II inhibition on varying stiffness recapitulates what is seen in normal and fibrotic lung. Normal human lung fibroblasts were fluorescently labeled and allowed to migrate on 1 and 25 kPa polyacrylamide gels (A and B; 6 h at 37 °C, ± 10 μm blebbistatin) or allowed to attach for 1 h then incubated in the presence of indicated reagents (C and D; 24 h at 37 °C). Panel A, comparison of the effect of blebbistatin and varying stiffness on number of protrusions (*, p < 0.001 versus 1 kPa vehicle only; +, p = 0.01 versus 25 kPa vehicle only. Panel B, comparison of the effect of blebbistatin and varying stiffness on cell polarity (*, p < 0.001 versus 1 kPa vehicle only; +, p = 0.001 versus 25 kPa vehicle only). 90 cells were analyzed for each condition in three separate experiments. Panel C, comparison of the effects of TGF-β and blebbistatin on actin density (+, p = 0.01 versus 25 kPa control; *, p < 0.001 versus 25 kPa TGF only, p < 0.001 by ANOVA). Panel D, comparison of the effects of TGF-β and blebbistatin on α-SMA density (*, p = 0.01 versus 25 kPa TGF only; +, p < 0.001 versus 1 kPa control by t test; p < 0.001 by ANOVA). 10 cells were analyzed for each condition in three separate experiments. Panel E, wind rose plots illustrating the differences in baseline migration characteristics of HLFs on PA gels versus lung tissue, and similarities in the effect of blebbistatin on migration (6 h, 10 μm blebbistatin).