Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1 , ROCK2 , and RhoA mRNAs were compared between each experiment. Top ROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (* p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing, Control, Negative Control, Western Blot, Incubation

Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Migration of TE-10 cell sheets with siRNA knockdown of target mRNAs 72 h after scraping. a Cell migration was quantified over a 48-h period. For each experiment, negative control values were used as the reference. The upper graphs show the migration velocity of the leading edge (Δ LE), while the lower graphs show the velocity of the leading line of the stratified region. Data shown represent the mean ± SEM of at least six independent experiments in which at least three series were stained with Hoechst 33342 and at least 3 series were stained with H2B-GFP. * p < 0.001; ** p = 0.002; *** p = 0.0028; **** p = 0.0044. ANOVA was performed using the original data. NC negative control. b Images of the cell sheets 72 h after scraping under siRNA treatment. First row negative control scrambled siRNA. The shape of the cells in the front row was very similar to that of untreated cells. There was no obvious irregularity in the arrangement of the cells. Second row knockdown of ROCKs by siRNA (ROCK1-#1 and ROCK2-#1). The arrangement of the cells in the leading row was obviously disordered. Many cells in the leading row were small with hypoplastic stress fibers. Several empty spaces between the cells were visible. Third row knockdown of RhoA by siRNA (RhoA-#1). The cells in the simple layer region were smaller than those in the negative control. Many of the cells were small and round, with inconspicuous stress fibers. Green α-tubulin, red β-actin, blue nuclei; scale bar 100 µm. Each inset shows the magnified image of the area surrounded by the interrupted white line on the merged image

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Migration, Knockdown, Negative Control, Staining

Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Sequences of chimeric siRNAs used in this study

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Negative Control

Journal: JCI Insight

Article Title: ER stress and Rho kinase activation underlie the vasculopathy of CADASIL

doi: 10.1172/jci.insight.131344

Figure Lengend Snippet: ( A ) VSMC Rho kinase activity assessed by ELISA ( n = 4–5; Student’s t test). ( B and C ) Expression of ROCK2 ( B ) and ROCK1 ( C ) in CADASIL and control VSMCs in the absence and presence of a γ-secretase inhibitor (GSI). Upper panels: Representative immunoblots. Lower panels: Quantification of ROCK2 and ROCK1 protein expression normalized to α-tubulin ( n = 6–10; two-way ANOVA with Bonferroni’s post hoc test). ( D ) Calcium transients were measured by live cell fluorescence imaging using the fluoroprobe Cal-520 AM. Representative tracings of VSMC [Ca 2+ ] i responses to Ang II (1 × 10 –7 mol/L) in CADASIL and control groups. Experiments were repeated 4 times/group with greater than 30 cells studied/field. ( E ) [Ca 2+ ] i calculated as the area under the curve. Arrow indicates time of Ang II stimulation ( n = 4; Student’s t test). Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 versus control; # P < 0.05 versus vehicle plus CADASIL.

Article Snippet: Antibodies used were as follows: anti-ROCK2 (1:500, 610623, BD Biosciences); anti-ROCK1 (1:500; PIPA521130, Chemicon International); anti-PCNA (sc-56, Santa Cruz Biotechnology); anti-BiP (1:1000; 610978, BD Biosciences); anti-Notch3 (1:2000; 5276, Cell Signaling Technology); anti–β-actin (1:5000; A1978, Sigma-Aldrich); anti–α-tubulin (1:10000, ab4074, Abcam); anti–phospho-cofilin (3313S, Cell Signaling Technology); anti–phospho-vimentin (7391S, Cell Signaling Technology); anti-vimentin (3632S, Cell Signaling Technology); anti–phospho-filamin (4761S, Cell Signaling Technology); anti-filamin (4762S, Cell Signaling Technology); and anti-Nox5 (1: 1000; provided by David Harrison, Vanderbilt University Medical Center, Nashville, Tennessee, USA) ( ).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Fluorescence, Imaging

Journal: JCI Insight

Article Title: ER stress and Rho kinase activation underlie the vasculopathy of CADASIL

doi: 10.1172/jci.insight.131344

Figure Lengend Snippet: ( A – D ) Pretreatment of VSMCs with 4-PBA (1 × 10 –3 mol/L) or fasudil (1 × 10 –5 mol/L) reduced expression of ( A ) HEYL and ( B ) HES5 , and ER stress–related genes ( C ) AMFR6 and ( D ) EDEM1 in CADASIL VSMCs without effect on control VSMCs ( n = 3–14; one-way ANOVA with Bonferroni’s post hoc test). ( E ) Increased expression of BiP in CADASIL VMSCs was reduced in 4-PBA (1 × 10 –3 mol/L) and fasudil (1 × 10 –5 mol/L) pretreated cells. Upper panel: Representative immunoblot of BiP. BiP expression was normalized to β-actin ( n = 4–10; one-way ANOVA with Bonferroni’s post hoc test). ( F ) 4-PBA decreased Rho kinase activity in CADASIL VMSCs, as assessed by ELISA ( n = 4–12; one-way ANOVA with Bonferroni’s post hoc test). Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.005 versus vehicle group from control VSMCs.

Article Snippet: Antibodies used were as follows: anti-ROCK2 (1:500, 610623, BD Biosciences); anti-ROCK1 (1:500; PIPA521130, Chemicon International); anti-PCNA (sc-56, Santa Cruz Biotechnology); anti-BiP (1:1000; 610978, BD Biosciences); anti-Notch3 (1:2000; 5276, Cell Signaling Technology); anti–β-actin (1:5000; A1978, Sigma-Aldrich); anti–α-tubulin (1:10000, ab4074, Abcam); anti–phospho-cofilin (3313S, Cell Signaling Technology); anti–phospho-vimentin (7391S, Cell Signaling Technology); anti-vimentin (3632S, Cell Signaling Technology); anti–phospho-filamin (4761S, Cell Signaling Technology); anti-filamin (4762S, Cell Signaling Technology); and anti-Nox5 (1: 1000; provided by David Harrison, Vanderbilt University Medical Center, Nashville, Tennessee, USA) ( ).

Techniques: Expressing, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex

doi: 10.1016/j.isci.2023.106240

Figure Lengend Snippet:

Article Snippet: Rabbit anti p-ROCK2 , GeneTex , Cat. # GTX122651; RRID: AB_2560946.

Techniques: Virus, Plasmid Preparation, Recombinant, Software

Journal: PLoS ONE

Article Title: Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells

doi: 10.1371/journal.pone.0226406

Figure Lengend Snippet: (A) Representative western blots showing the effects of ROCK1 or ZIPK knockdown on ROCK1 and ZIPK expression in CASMC and UASMC. (B) Quantification of ROCK1, ROCK2 and ZIPK levels in ROCK1 siRNA-treated cells compared to controls. (C) Quantification of ROCK1 and ZIPK levels in ZIPK siRNA-treated cells compared to controls. Statistical analysis was carried out with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used in this study: rabbit monoclonal anti-ZIPK (1:1,000 dilution; Epitomics, Burlingame, CA; cat. no. 2568–1), rabbit polyclonal anti-ROCK1 (1:1,000 dilution; Epitomics; cat. no. S2711), rabbit polyclonal anti-ROCK2 (1:2,000 dilution; AbFrontier, Seoul, South Korea; cat. no. LF-PA0049), rabbit polyclonal anti-pT18/pS19-myosin light chain 2 (1:500 dilution; Cell Signaling; cat. no. 3674), rabbit anti-GAPDH (1:5,000 dilution; Santa Cruz Biotechnology; cat. no. sc-32233), goat polyclonal anti-SM22α (1:2,500 dilution; Novus Biologicals, Centennial, CO; cat. no. NB600-507), rabbit polyclonal anti-LC 20 (1:2,000 dilution; Rockland Immunochemicals, Limerick, PA; cat. no. 600-401-938), rabbit polyclonal anti-pT853-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. 36–003), rabbit polyclonal anti-pT696-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. ABS45), rabbit polyclonal anti-pT163-Par4 (1:1,000 dilution; Cell Signaling; cat. no. 2329), rabbit monoclonal anti-pS473-Akt (193H12) (1:1,000 dilution; Cell Signaling; cat. no. 4058); rabbit polyclonal anti-pT180/pY182-p38 MAP kinase (1:1,000 dilution; Cell Signaling; cat. no. 9211), rabbit monoclonal anti-pT202/Y204-ERK1/pT185/pY187-ERK2 (1:1,000 dilution; Cell Signaling; cat. no. 4370), rabbit anti-pS82-HSP27 (1:1,000 dilution; Cell Signaling; cat. no. 2401), and rabbit polyclonal anti-α-actin (1:1,000 dilution; Cytoskeleton, Inc.; cat. no. AAN01).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells

doi: 10.1371/journal.pone.0226406

Figure Lengend Snippet: A schematic showing the main conclusions from this study regarding serum-induced phosphorylation of the regulatory light chains of myosin II (LC 20 ) and the regulatory kinases and phosphatase involved. Serum treatment of CASMC and UASMC activates Rho-associated coiled-coil kinase (ROCK) and zipper-interacting protein kinase (ZIPK) leading to phosphorylation of the regulatory myosin-targeting subunit (MYPT1) of myosin light chain phosphatase (MLCP) at T853 and prostate apoptosis response 4 (Par4) at T163, both of which result in inhibition of MLCP activity. ZIPK activation involves phosphorylation by ROCK and autophosphorylation. Activated ROCK and ZIPK directly phosphorylate LC 20 at T18 and S19.

Article Snippet: The following primary antibodies were used in this study: rabbit monoclonal anti-ZIPK (1:1,000 dilution; Epitomics, Burlingame, CA; cat. no. 2568–1), rabbit polyclonal anti-ROCK1 (1:1,000 dilution; Epitomics; cat. no. S2711), rabbit polyclonal anti-ROCK2 (1:2,000 dilution; AbFrontier, Seoul, South Korea; cat. no. LF-PA0049), rabbit polyclonal anti-pT18/pS19-myosin light chain 2 (1:500 dilution; Cell Signaling; cat. no. 3674), rabbit anti-GAPDH (1:5,000 dilution; Santa Cruz Biotechnology; cat. no. sc-32233), goat polyclonal anti-SM22α (1:2,500 dilution; Novus Biologicals, Centennial, CO; cat. no. NB600-507), rabbit polyclonal anti-LC 20 (1:2,000 dilution; Rockland Immunochemicals, Limerick, PA; cat. no. 600-401-938), rabbit polyclonal anti-pT853-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. 36–003), rabbit polyclonal anti-pT696-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. ABS45), rabbit polyclonal anti-pT163-Par4 (1:1,000 dilution; Cell Signaling; cat. no. 2329), rabbit monoclonal anti-pS473-Akt (193H12) (1:1,000 dilution; Cell Signaling; cat. no. 4058); rabbit polyclonal anti-pT180/pY182-p38 MAP kinase (1:1,000 dilution; Cell Signaling; cat. no. 9211), rabbit monoclonal anti-pT202/Y204-ERK1/pT185/pY187-ERK2 (1:1,000 dilution; Cell Signaling; cat. no. 4370), rabbit anti-pS82-HSP27 (1:1,000 dilution; Cell Signaling; cat. no. 2401), and rabbit polyclonal anti-α-actin (1:1,000 dilution; Cytoskeleton, Inc.; cat. no. AAN01).

Techniques: Inhibition, Activity Assay, Activation Assay