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: 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: Pharmaceuticals

Article Title: 9-Methylfascaplysin, a Marine-Derived Bioactive Compound, Promotes Neurite Outgrowth via the Inhibition of ROCK2

doi: 10.3390/ph18111751

Figure Lengend Snippet: 9-MF induces neurite outgrowth via the action on a ROCK2-centralized network. ( A ) Volcano plot showing RNA-seq results from 9-MF- and vehicle-treated PC12 cells. ( B ) GO-CC analysis of 9-MF-associated DEGs showing the enrichment in mitochondria and synapse. ( C ) KEGG analysis of 9-MF-associated DEGs showing enrichment in various neurodegenerative disorders. ( D ) The PPI network of 9-MF-associated DEGs showing a ROCK2-centralized network. ( E ) Venn diagram showing the intersection of 9-MF-associated DEGs and neurite-outgrowth-related genes. ( F ) GO-MF of DEGs in ( E ) showing the enrichment in kinase activity and microtubule binding. ( G ) Venn diagram showing the intersection of 9-MF-associated DEGs and mitochondrial metabolism-related genes. ( H ) GO-MF of DEGs in ( G ) showing the enrichment of DEGs in GDP-dissociated inhibitor binding and GTPase motor activity.

Article Snippet: The membranes were blocked with 5% bovine serum albumin in tris-buffered saline with Tween-20 (TBST) for 2 h and incubated overnight at 4 °C with primary antibodies [GAPDH (1:2000), GAP-43 (1:2000), ROCK2 (1:2000), or p-ROCK2 (Ser1366, 1:2000) were from Affinity Biosciences, Cincinnati, OH, USA].

Techniques: RNA Sequencing, Activity Assay, Binding Assay

Journal: Pharmaceuticals

Article Title: 9-Methylfascaplysin, a Marine-Derived Bioactive Compound, Promotes Neurite Outgrowth via the Inhibition of ROCK2

doi: 10.3390/ph18111751

Figure Lengend Snippet: 9-MF inhibits ROCK2. ( A ) CMap analysis revealed that the transcriptional response to 9-MF positively correlated with ROCK2 pathway inhibition, including profiles induced by LIMK2 knockdown and Rho GTPase-activating protein overexpression. Conversely, 9-MF negatively correlated with perturbations associated with Rho pathway activation. ( B ) Dot plot showing the affinities between various ROCK2 inhibitors and ROCK2. The size and color of the dots correspond to the affinity. The specific affinities between ROCK2 and inhibitors are as follows: 9-MF (−10.0 kcal/mol), zelasudil (−9.41 kcal/mol), belumosudil (−9.38 kcal/mol), hydroxyfasudil (−8.22 kcal/mol), ripasudil (−8.06 kcal/mol), azaindole-1 (−7.84 kcal/mol), fasudil (−7.81 kcal/mol), and Y-27632 (−7.07 kcal/mol). ( C ) The best conformation between 9-MF and ROCK2 was elucidated by molecular docking analysis.

Article Snippet: The membranes were blocked with 5% bovine serum albumin in tris-buffered saline with Tween-20 (TBST) for 2 h and incubated overnight at 4 °C with primary antibodies [GAPDH (1:2000), GAP-43 (1:2000), ROCK2 (1:2000), or p-ROCK2 (Ser1366, 1:2000) were from Affinity Biosciences, Cincinnati, OH, USA].

Techniques: Inhibition, Knockdown, Over Expression, Activation Assay

Journal: Pharmaceuticals

Article Title: 9-Methylfascaplysin, a Marine-Derived Bioactive Compound, Promotes Neurite Outgrowth via the Inhibition of ROCK2

doi: 10.3390/ph18111751

Figure Lengend Snippet: 9-MF induces neurite outgrowth via the inhibition of ROCK2. 9-MF and fasudil were added into PC12 cells for 0.5 h. ( A ) Immunocytochemical staining was employed to measure p-ROCK2-positive area. ( B ) The quantitative results of ( A ) showed that 9-MF upregulated the p-ROCK2-positive area in PC12 cells (n = 10). ( C ) Western blotting analysis was employed to measure the expression of p-ROCK2 and ROCK2 (n = 3). 9-MF and narciclasine were added into PC12 cells for 48 h. ( D ) FDA staining was employed to measure neurite outgrowth. ( E ) The quantitative results of ( D ) showed that 9-MF induced neurite outgrowth in PC12 cells (n = 10). Narc: narciclasine. Data were presented as the mean ± SD. * p < 0.05 and ** p < 0.01 vs. the control group in ( B , C ) (one-way ANOVA and Tukey’s test), ** p < 0.01 vs. the control group, and # p < 0.05 vs. the 9-MF group in ( E ) (two-way ANOVA and Tukey’s test).

Article Snippet: The membranes were blocked with 5% bovine serum albumin in tris-buffered saline with Tween-20 (TBST) for 2 h and incubated overnight at 4 °C with primary antibodies [GAPDH (1:2000), GAP-43 (1:2000), ROCK2 (1:2000), or p-ROCK2 (Ser1366, 1:2000) were from Affinity Biosciences, Cincinnati, OH, USA].

Techniques: Inhibition, Staining, Western Blot, Expressing, Control

Journal: Cancer Cell International

Article Title: LncRNA ZFAS1 promotes pancreatic adenocarcinoma metastasis via the RHOA/ROCK2 pathway by sponging miR-3924

doi: 10.1186/s12935-020-01322-8

Figure Lengend Snippet: ROCK2 functions as the target of ZFAS1/miR-3924. a The differentially expressed genes in the high ZFAS1 expression group were focused primarily in the focal adhesion pathway. b The Venn diagram shows the target mRNAs predicted with GSEA, TargetScan 7.2 and miRDB. c The binding site of miR-3924/ROCK2 was predicted with TargetScan 7.2. d 293T cells were transfected with ROCK2 3′-UTR Wt, Mut, NC or PC reporter plasmids along with miR-3924 mimics, and relative luciferase activity was measured (n = 3), *p < 0.05. e – h Western blot assays showed that the expression levels of FAK, RHOA and ROCK2 in the sh-ZFAS1 and miR-3924 mimic groups were lower than those in the mock group (n = 3), *p < 0.05

Article Snippet: The primary antibodies were as follows: anti-ROCK2 (ab125025, Abcam, USA), anti-FAK (ab40794, Abcam, USA), anti-RHOA (ab187027, Abcam, USA) and anti-β-ACTIN (ab8227, Abcam, USA).

Techniques: Expressing, Binding Assay, Transfection, Luciferase, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Decompensated liver cirrhosis and neural regulation of mesenteric vascular tone in rats: role of sympathetic, nitrergic and sensory innervations

doi: 10.1038/srep31076

Figure Lengend Snippet: ( A ) Effect of liver cholestasis on alpha 1- adrenoceptor and alpha 2B- adrenoceptor expression. The blot is representative of six separate segments from each group. Rat brain homogenates were used as a positive control. Lower panel shows relation between alpha 1-adrenoceptor or alpha 2B-adrenoceptor expression and β-actin. Results (mean ± SEM) are expressed as a ratio of the signal obtained for each protein and the signal obtained for β-actin. *P < 0.05 SO vs. LC rats (Student’s t-test). ( B ) Effect of liver cholestasis on ROCK1, ROCK2, P-MYPT and MYPT expression. The blot is representative of six separate segments from each group. HeLa cell lysates were used as a positive control. Lower panel shows relation between ROCK1, ROCK2, P-MYPT or MYPT expression and β-actin. Results (mean ± SEM) are expressed as a ratio of the signal obtained for each protein and the signal obtained for β-actin. *P < 0.05 SO vs. LC rats (Student’s t-test).

Article Snippet: For these experiments, we used a mouse monoclonal antibody against nNOS (1:2000, BD Biosciences), a rabbit polyclonal antibody against nNOS phosphorylated In Ser1417 (P-nNOS, 1:2000, Abcam), a rabbit monoclonal antibody against Rho kinase 1 (ROCK1, 1:1000, Abcam), a mouse monoclonal antibody against ROCK2 (1:500, Abcam), a rabbit polyclonal antibody against myosin phosphatase (MYPT, 1:2000, Abcam), a rabbit polyclonal antibody against MYPT phosphorylated In Thr696 (P-MYPT, 1:500, Abcam), and a monoclonal anti-β-actin-peroxidase antibody (1:50000, Sigma-Aldrich, Spain).

Techniques: Expressing, Positive Control

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Schematic representation of mouse ROCK1 and 2 proteins, Rock1 and 2 loci, targeted and deleted alleles. ( B ) Proliferation curves of Rock1 f/f ;Rock2 f/f control, Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs 11 to 16 days after seeding. Cells were seeded 3 days after adenovirus infection. Graph shows total number of cells and SD from three independent experiments, each carried out in triplicates. ( C ) Cells were treated with Y-27632 and H1152. One day later, equal numbers of cells were plated and subjected to growth analysis. The graph shows average number of cells and SD from at least 3 independent experiments, each carried out in triplicates. DOI: http://dx.doi.org/10.7554/eLife.12203.004

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Proliferation curves of MEFs with different genotypes over 6 days. The cells were seeded 3 d after adenovirus infection. Graphs show total number of cells and SD from 5 independent experiments each carried out in triplicates. p-values were calculated using Student’s t-test: ** p<0.005; *** p<0.001. ( B ) Rock1 f/f ;Rock2 f/f control and Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs were cultured for 3 days and wild-type cells were treated with H1152, inactive blebbistatin (+) or active blebbistatin (+/-) for 48 hr. Cells from all conditions were then subjected to a colony formation assay and grown for a further 7 days. ( C–F ) Rock1 f/f ;Rock2 f/f MEFs transformed with Trp53 DD and HRas V12 were treated with Ad Cre to generate ∆. Cells were injected subcutaneously into CD1 nude mice and growth analyzed. The graph shows average tumor volume in mm 3 and SEM for Rock1 ∆/∆ and control ( C ), Rock2 ∆/∆ and control ( D ), Rock1 ∆/∆ ;Rock2 ∆/∆ and control ( E ). p values were calculated by ANOVA and are as indicated. ( F ) Tumors with stated genotypes were immunoblotted with indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.12203.003

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection, Cell Culture, Colony Assay, Transformation Assay, Injection

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Western blot analysis of ROCK1 and 2 in Rock1 f/f ;Rock2 f/f cells after indicated days of Ad-GFP and Ad-Cre-GFP infection. ( B ) Cells from time-lapse movies were tracked and their migration speed and directionality determined using using ImageJ analysis software. Data are from five independent experiments and p-values were calculated using Student’s t-test: ** p<0.01. ( C ) Quantification of western blot analyses. Graphs show protein levels of ROCK1, ROCK2 divided by total ERK protein levels and pMLC, pMYPT850, pMYPT696 and pCofilin divided by their total protein levels and SEM. The data are from five independent experiments and p-values were calculated using Student’s t-test: *** p<0.001. ( D ) MEFs were treated with H1152 for 20 min and the phospho-proteome was analyzed by quantitative mass spectrometry. The graph shows log ratios of identified phospho-peptides from two reciprocally heavy vs. light SILAC-labelled control and H1152-treated cell populations. The ‘Significant-B’ outlier test was used to determine significantly regulated peptides in both replicates, using a Benjamini-Hochberg FDR rate of 5%. ( E ) MEFs were treated with H1152 overnight and the phospho-proteome was analyzed by quantitative mass spectrometry. The graph shows log ratios of identified phospho-peptides from two reciprocally heavy vs. light SILAC-labelled control and H1152-treated cell populations. The ‘Significant-B’ outlier test was used to determine significantly regulated peptides in both replicates, using a Benjamini-Hochberg FDR rate of 5%. DOI: http://dx.doi.org/10.7554/eLife.12203.006

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Western Blot, Infection, Migration, Software, Mass Spectrometry

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Images of wild-type, Rock1 ∆/∆ , Rock2 ∆/∆ or Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs, 3 days after Ad-GFP and Ad-Cre-GFP infection, stained for pMLC and phalloidin. Wild-type and Rock1 ∆/∆ ;Rock2 ∆/∆ cells are also shown 5 days after Ad-GFP and Ad-Cre-GFP infection. Scale bars are 50 µm. ( B, C ) Western blot analyses of ROCK targets in lysates of MEFs with indicated genotypes. Representative blots are shown, quantification of multiple biological replicates can be found in . ( D ) Images of MEFs with indicated genotypes plated on a thick layer of collagen and stained for phalloidin. Scale bars are 50 µm. ( E ) Images and quantification of collagen gel contraction assay using MEFs of indicated genotypes. Graph shows average data and SD from 5 independent experiments, each carried out in triplicate. p-values were calculated using Student’s t-test: ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.12203.005

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection, Staining, Western Blot, Collagen Gel Contraction Assay

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Images of MEFs treated with H1152, blebbistatin (+), blebbistatin (+/-) for 5 days and Rock1 f/f ;Rock2 f/f control , Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs, 5 days after Ad-GFP and Ad-Cre-GFP infection and followed by SA-βgal staining. Scale bars are 50 µm. Graph shows number of SA-βgal expressing cells divided by total number of cells and SD of >100 cells from three independent experiments and p-values were calculated using Student’s t-test: *** p<0.001. ( B ) Images of Rock1 f/f ;Rock2 f/f and Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs stained with phalloidin and DAPI. Overlay images shown. Arrows in image indicate binucleate cells. Scale bars are 50 µm. Bar chart shows average data and SD of nuclei in > 150 cells from 5 independent experiments. Values were calculated using Student’s t-test: * p<0.05. ( C, D ) Analysis of cell division of Rock1 f/f ;Rock2 f/f and Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs in time-lapse movies. ( C ) Quantification of failed cell divisions resulting in binucleate cells. ( D ) Quantification of average number of cells dividing. Graphs show average data and SD of > 300 cells from at least five independent experiments. ( E ) Cell cycle profiles of Rock1 f/f ;Rock2 f/f and Rock1 ∆/∆ ;Rock2 ∆/∆ MEFs. The graph shows the percentage of cells in G2/M (top), S (middle) and G1 (bottom) phase of the cell cycle. Error bars represent SD. Data are from 5 independent experiments and p-values were calculated using Student’s t-test: * p<0.05. DOI: http://dx.doi.org/10.7554/eLife.12203.009

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection, Staining, Expressing

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Melanoma lines were treated with the indicated inhibitors for 7 days and subjected to SA-βgal staining. Scale bars are 50 µm. ( B, C ) Intra-dermal injection of 690cl2 melanoma cells into CD1 athymic mice followed by treatment with AT13148 (40 mg/kg) or vehicle. The graph shows average tumor volume in mm 3 and SEM ( B ). Values calculated by Student’s t-test are as indicated. The lungs were analyzed by SA-βGal staining. The graph shows the number of SA-βGal-positive cells per 10 fields of view ( C ). p-values calculated by ANOVA are as indicated. ( D ) Rock1 f/f ;Rock2 f/f and Rock1 ∆/∆ ;Rock2 ∆/∆ cells treated with nocodazole (4 µg/ml) or vehicle for 48 hrs were subjected to propidium iodide staining and flow cytometry analysis. The graph shows the percentage of cells in G2/M (top), S (middle) and G1 (bottom) phase of the cell cycle. Data are from three independent experiments and error bars represent SD. DOI: http://dx.doi.org/10.7554/eLife.12203.010

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Staining, Injection, Flow Cytometry

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) Rock1 f/f ;Rock2 f/f cells, immortalized with SV40 Large T, were infected with Ad-GFP or Ad-Cre-GFP to generate Rock1 f/f ;Rock2 f/f control and Rock1 ∆/∆ ;Rock2 ∆/∆ cells. These were subjected to growth analysis. The graph shows average data and SD from 3 independent experiments, each carried out in triplicates. ( B ) Rock1 f/f ;Rock2 f/f ;Trp53 f/f and Rock1 ∆/∆ ;Rock2 ∆/∆ ;Trp53 ∆/∆ cells were seeded at equal numbers and kept for ~7 days allowing colonies to form. In parallel, cells were subjected to SA-βgal staining and western blot analysis using indicated antibodies. Scale bars in images are 50 µm. ( C ) Trp53 wild-type and Trp53 null lines were treated with the indicated inhibitors for 7 days and subjected to SA-βgal staining. Scale bars are 100 µm. DOI: http://dx.doi.org/10.7554/eLife.12203.014

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection, Staining, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) MEFs were treated with H1152 or vehicle for 3 days and the proteome analyzed by quantitative mass spectrometry. Graph shows log ratios of identified proteins. The data are from duplicate experiments of reciprocal SILAC labelling. Proteins indicated are Cdkn2a/Arf (p19), Cdkn2a/Ink4a (p16), Cdkn2b (p15), Ccnd2 (CyclinD2), Ccnd1 (CyclinD1), Ccnd3 (CyclinD3), Cdk2, Cdk4, Cdkn1b (p27), Ccnb1 (CyclinB1). ( B ) Rock1 f/f , Rock2 f/f , Rock1 f/f ;Rock2 f/f and where indicated wild type cells infected with Ad-Cre-GFP were subjected to immunoblotting with antibodies against proteins indicated. DOI: http://dx.doi.org/10.7554/eLife.12203.015

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Mass Spectrometry, Infection, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A ) MEFs were treated with H1152 or vehicle for 3 days and the proteome analyzed by quantitative mass spectrometry. Graph shows log ratios of identified proteins. Data are from duplicate experiments of reciprocal SILAC labelling. The ‘Significant-B’ outlier test was used to determine significantly regulated peptides or proteins, using a Benjamini-Hochberg FDR rate of 5%. ( B, C ) MEFs of different genotypes or treated with indicated inhibitors were immunoblotted for pCDK1, total CDK1, Cyclin A, CKS1, ROCK1, ROCK2 and total ERK as loading control ( B ). ( C ) Quantification of western blot analyses. Graphs show protein levels of CDK1, CyclinA and CKS1 divided by total ERK protein levels and SEM in indicated samples. The data are from 5 independent experiments and p-values were calculated using Student’s t-test: * p<0.05, ** p<0.005, *** p<0.001. ( D ) qPCR analysis of mRNA levels of Cks1b, Cdk1 and Ccna2 in indicated samples. Graphs show average normalized mRNA levels and SD from at least five independent experiments, each carried out in triplicates. p-values were calculated using Student’s t-test: * p<0.05, *** p<0.001. DOI: http://dx.doi.org/10.7554/eLife.12203.013

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Mass Spectrometry, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A–C ) MEFs were infected with either control shRNA or shRNAs targeting Cks1b, Cdk1 and Ccna2. Four days after infection, equal numbers of cells were plated and kept for seven days, allowing colonies to form ( A ). Five days after infection with shRNAs, cells were also subjected to SA-βGal staining ( B ) and immunoblotting with antibodies against pCDK1, total CDK1, Cyclin A, CKS1. ROCK2 and total ERK were used as loading control ( C ). Scale bars are 50 µm. DOI: http://dx.doi.org/10.7554/eLife.12203.016

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Infection, shRNA, Staining, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: Cohorts of mice carrying LSL-Kras G12D ; LSL-Trp53 R270H alleles and the indicated Rock alleles were treated once with Ad-Cre viruses by intranasal inhalation. 24 weeks later, the mice were sacrificed and the lungs were analyzed by histopathology. ( A ) Graphs show tumor area divided by lung area for indicated genotypes and tumor grades. The data are also displayed as a sum of tumor area divided by lung area for the indicated genotypes. Graphs show average data and SEM. The total number of mice analyzed is indicated in Scatter Plots. ( B ) Cell lines derived from Kras G12D ;Trp53 R270H tumors of either Rock1 f/f , Rock2 f/f , Rock1 f/f ;Rock2 f/f or wild-type genotype were subjected to immunoblotting with indicated antibodies. ( C ) Isolated lung tumor cell lines of Rock1 ∆/wt ;Rock2 ∆/∆ genotype as well as a cell line which retained both Rock1 alleles ( Rock1 f/f ;Rock2 ∆/∆ ) were treated with Ad-GFP and Ad-Cre-GFP. 3 days after infection, cells were plated and kept for ~5 days allowing colonies to form. ( D ) Absolute quantification of ROCK1 and 2, using selective reaction monitoring (SRM). Bar graphs show the mean concentration in nM of ROCK1 (black) and ROCK2 (grey) across at least 3 biological replicates from each genetic background. Line 1–3 indicate different cell lines isolated from NSCLCs and Tumors 1–4 represent different micro-dissected tumors. Error bars represent SEM. DOI: http://dx.doi.org/10.7554/eLife.12203.017

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Histopathology, Derivative Assay, Western Blot, Isolation, Infection, Concentration Assay

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/eLife.12203

Figure Lengend Snippet: ( A, B ) Scatter plots showing time until melanomas developed in mice carrying Tyr-Cre ERT2 ; Braf V600E alleles and where indicated Pten f/wt , Pten f/f and Rock alleles ( A ) as well as Kaplan-Meier survival curves ( B ). The same number of mice indicated in scatter plots was analyzed in survival curve. ( C ) Absolute quantification of ROCK1 and 2, using selective reaction monitoring (SRM). Bar graphs show the mean concentration in nM of ROCK1 (black) and ROCK2 (grey) across at least 3 biological replicates of different cell lines isolated from mice with a Tyr-Cre ERT2 ; Braf V600E ; Pten f/wt genetic background with Rock alleles as indicated. Error bars represent SEM. ( D ) Immunoblotting of lysates from melanoma cell lines derived from tumors with ROCK1 and 2 antibodies. GAPDH or ERK were used as loading control. ( E ) Melanoma cell lines of Rock2 ∆/∆ genotype as well as a cell line which retained both Rock2 alleles ( Rock1 ∆/∆ ;Rock2 f/f ) were treated with Ad-GFP and Ad-Cre-GFP to generate controls, Rock2 ∆/∆ and Rock1 ∆/∆ ;Rock2 ∆/∆ cells. 3 days after infection, the cells were plated and kept for ~5 days allowing colonies to form. DOI: http://dx.doi.org/10.7554/eLife.12203.018

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phospho-p44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKα) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house ( ).

Techniques: Concentration Assay, Isolation, Western Blot, Derivative Assay, Infection