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.