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: Molecular neurobiology

Article Title: RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA 2 Activation.

doi: 10.1007/s12035-016-0187-6

Figure Lengend Snippet: Fig. 2 TNF-α and glutamate induced activation of RhoA, Rho kinase and cPLA2. a, b Western blot analysis showed that the activation of RhoA (a), Rho kinase and cPLA2 (b) were significantly increased. c, d Densitometric quantification of RhoA, Rho kinase, and cPLA2 activation. Data were expressed as fold change compared to the control group. Values are means ± s.e.m. (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Briefly, spinal cord neurons on coverslips on day 7 were fixed by 4%PFA followed bywashingwith 0.01M PBS, blocking 1 h with 5 % goat serum-0.3 % Triton X-100PBS, and staining with anti-rabbit βIII tubulin(1:1000, Cell Signaling, USA), anti-mouse NeuN (1:100, EMD Millipore, USA), anti-rabbit ROCK1 (1:100) or anti-rabbit ROCK2 (1:100, Santa Cruz, USA) [27], anti-mouse cPLA2 (1:50, Santa Cruz, USA) antibodies at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Control

Journal: Molecular neurobiology

Article Title: RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA 2 Activation.

doi: 10.1007/s12035-016-0187-6

Figure Lengend Snippet: Fig. 7 Schematic drawing illustrates the proposed mechanism of RhoA/Rho kinase and cPLA2 activation following spinal cord injury. The combined TNF-α and glutamate insults activate RhoA/Rho kinase and cPLA2. The phospho-cPLA2 re-localizes to cell and organelle membrane to hydrolyze phospholipids to arachidonic acid (AA) and eicosanoids to induce neuronal death. Inhibition of RhoA, ROCK1/2, and cPLA2 with CT04, Y27632, and ATK reduces cPLA2 activation and neuronal death

Article Snippet: Briefly, spinal cord neurons on coverslips on day 7 were fixed by 4%PFA followed bywashingwith 0.01M PBS, blocking 1 h with 5 % goat serum-0.3 % Triton X-100PBS, and staining with anti-rabbit βIII tubulin(1:1000, Cell Signaling, USA), anti-mouse NeuN (1:100, EMD Millipore, USA), anti-rabbit ROCK1 (1:100) or anti-rabbit ROCK2 (1:100, Santa Cruz, USA) [27], anti-mouse cPLA2 (1:50, Santa Cruz, USA) antibodies at 4 °C overnight.

Techniques: Activation Assay, Membrane, Inhibition