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. 2008 Oct 31;283(44):29888–29896. doi: 10.1074/jbc.M803880200

FIGURE 4.

FIGURE 4.

ROCK and PRK control distinct thrombin-induced contractility pathways. a, human endothelial cells were transfected with 50 nm nonsilencing RNA (nsi) or two independent sequences targeting PRK (si#1 and si#2) for 3 days. Overnight starved cells were left unstimulated or simulated with thrombin (0.1 unit/ml, 5 min), and total cell lysates were used for Western blot against phospho-specific (p) MLC, cofilin (Cof), paxillin (Pax), and FAK. siRNA knockdown was checked by PRK Western blot, and tubulin was used as a loading control. b, similar experiments were performed in endothelial cells transfected with mock DNA or Myc-tagged ROCK-KD, whose expression level was checked by Myc Western blots. c, levels of phosphorylation of MLC, cofilin, (Cof), paxillin (Pax), and FAK were estimated on scanned membranes (Licor) from multiple experiments and expressed as a normalized ratio on tubulin. d, nonsilencing-(nsi-), PRK siRNA-, mock-, and ROCK-KD-transfected human endothelial cells were grown on collagen-coated glass coverslips for 3 days as monolayers, starved overnight, and then stimulated with thrombin. Nontransfected cells were left unstimulated and used as control (Ctrl). Actin cytoskeleton (red) was revealed by phalloidin labeling and focal adhesions (green) by anti-paxillin immunostaining. Binary calculation (bin.) was applied on paxillin threshold pictures to unveil focal adhesion morphology (ImageJ software). e, number of cells presenting actin stress fibers was manually counted in 250 cells and expressed as the percentage of total cells in nonstimulated (ctrl) and nonsilencing-, PRK siRNA-, mock-, and ROCK-KD-transfected cells exposed to thrombin. f and g, similarly treated cells were analyzed by confocal microscopy and post-treated for binary processing on threshold pictures. Both the number of particles (f) and the average pixel size (g) were calculated for the paxillin-positive focal adhesions per field (n = 9). All panels are representative of at least three independent experiments. ANOVA test: *, p < 0.05; **, p < 0.01.