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. 2020 Jan 1;31(1):45–58. doi: 10.1091/mbc.E19-03-0131

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© 2020 McKenzie, Svec, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — Actomyosin contractility regulates leading-edge velocity and PKA activity. (A) A migrating SKOV-3 cell expressing pmAKAR3 was plated on a fibronectin-coated glass-bottomed imaging dish and monitored by live-cell microscopy before and after treatment with 25 µM blebbistatin (Blebb). Representative pseudocolored FRET/CFP images (corresponding to the boxed region in Supplemental Movie S3) are shown. (B) A maximum-intensity projection of cumulative PKA activity in 10 images (2 min apart) before and after treatment with blebbistatin. (C) Representative images of pmAKAR3-expressing SKOV-3 cells migrating on fibronectin-coated dishes before (0 min) and 30 min after treatment with 0.1% vol/vol dimethyl sulfoxide (DMSO), 1 µM H-1152, or 10 µM fasudil. The leading edge is magnified in the insets (scale bar = 10 µm). (D) QuimP11 software (Bosgraaf and Van Haastert, 2010) was used to generate maps of PKA activity (top) and edge velocity (bottom) within a 10-µm band along the leading edge before and after treatment with blebbistatin (Blebb). (E) SKOV-3 cells expressing mCherry–paxillin, mCherry–zyxin, or pmAKAR3 (FRET) migrating on fibronectin-coated glass dishes, or cells plated on 125 kPa fluorescent nanosphere-functionalized hydrogels (traction force) were treated with 25 µM blebbistatin (or with 0.1% vol/vol DMSO; Ctrl) at time = 0. Capturing images every 60 s, the fluorescence intensity of paxillin or zyxin within focal adhesions, PKA activity (via the pmAKAR FRET signal), or traction force was measured, normalized to values at t = 0, and plotted. The graph depicts mean values ± SEM (n_{paxillin-Blebb} = 211 adhesions from seven cells; n_zyxin-Blebb = 156 adhesions from five cells; n_FRET = 30 linescans from six cells; n_zyxin-Ctrl = 125 adhesions from five cells; n_FRET-Ctrl = 35 linescans from seven cells; n_{traction force} = average traction from six cells). The inset shows the first 5 min of the time course at higher resolution; these data were used to calculate the one-phase half-life (t_½) or apparent t_½ decay values for each signal, using GraphPad Prism.