Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: Silencing CD147 leads to decreased p190-B expression. a , b Relative quantitative real-time RT-PCR analysis of p190 RhoGAP family in indicated cells transfected with siCD147 or siCtrl at 24 h after transfection. ***p < 0.001, ns p > 0.05 by student’s t test. c , d The expression of p190-A or p190-B in indicated cells transfected with siCD147 or siCtrl was determined by Western blot at 48 h after transfection

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: Overexpression of CD147 results in upregulation of p190-B expression. a – c Relative quantitative real-time RT-PCR analysis of p190 RhoGAP family in indicated cells transfected with pCMV-HA or CD147-pCMV-HA at 24 h after transfection. ***p < 0.001, **p < 0.01, ns p > 0.05 by student’s t test. d The expression of indicated molecules in SMMC-7721 cells transfected with pCMV-HA or CD147-pCMV-HA was determined by Western blot at 48 h after transfection. Error bars indicate SD from at least triplicate determinations

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: Immunohistochemical analysis of CD147 and p190-B expression in the resected tumor. a – c Representative images of immunohistochemical staining for CD147 and p190-B in paired HCC tissues. The scores for CD147 in patient 040081, 062474 and 080532 were grade 1, grade 2 and grade 2, respectively. The scores for p190-B in the indicated patients were grade 2, grade 2 and grade 1, respectively. The scale bar represents 10 μm. d The relationship between CD147 and p190-B expression. **p = 0.001 by Spearman correlation

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: Correlation between CD147 and p190-B expression in HCC tissues

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Expressing

Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: p190-B promotes cell movement and inhibits RhoA activation. SMMC-7721 cells were transfected with a pool of siRNAs targeting p190-B or siCtrl. a Quantitative analysis of relative migration distance by wound-healing assay. ***p < 0.001 by student’s t test. b Quantification of RhoA activation in indicated cells at 48 h after transfection. *p < 0.05 by student’s t test. c Cells were stained with DAPI, rhodamine-conjugated phalloidin and an antibody against paxillin and imaged by confocal microscopy. Arrows indicate focal adhesions. Error bars indicate SD from at least triplicate determinations

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Activation Assay, Transfection, Migration, Wound Healing Assay, Staining, Confocal Microscopy

Journal: Cancer Cell International

Article Title: CD147 promotes cell motility via upregulation of p190-B RhoGAP in hepatocellular carcinoma

doi: 10.1186/s12935-016-0344-z

Figure Lengend Snippet: CD147 inhibits RhoA activation through enhancing p190-B expression. SMMC-7721 cells were transfected with the indicated constructs. a The expression of CD147 and p190-B was analyzed by Western blot. b Quantitative analysis of relative migration distance by wound-healing assay. *p < 0.05, ***p < 0.001 by ANOVA. c Schematic representation of major mechanisms of CD147 in regulating cancer cell movement via promoting p190-B expression. Error bars indicate SD from at least triplicate determinations

Article Snippet: CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen.

Techniques: Activation Assay, Expressing, Transfection, Construct, Western Blot, Migration, Wound Healing Assay

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: Engagement of syndecan-4 regulates membrane association of p190-A. Fibroblasts prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) before preparing lysates and measuring redistribution of proteins between soluble and membrane-bound fractions or RhoA activity by Western blotting. (A and B) Soluble, tyrosine-phosphorylated protein in lysates prepared in 0.5% (wt/vol) TrX or 1% (wt/vol) TrX, 0.5% (wt/vol) deoxycholate, and 0.1% (wt/vol) SDS. Arrowheads indicate a 190-kD band that varied in abundance between conditions. (C and D) TrX-soluble p190-A in lysates after stimulation with H/0 or a heparin-binding mutant (H/0-GAG). (E) Cytosolic and membrane-bound p190-A in sonication lysates. (F) Relative distribution of p190-A and cytosolic and membrane-bound markers, tailless complex polypeptide 1α and transferrin receptor, between membrane and cytosolic fractions of sonication lysates prepared from equal numbers of cells spread on 50K and stimulated with H/0 for 10 min. (G) Correlation between RhoA activity (dashed line) and membrane-associated p190-A (solid line). Equal loading between time points was confirmed by blotting for vinculin or HSP70. Error bars represent standard error. *, significant change (P < 0.05) from experiments repeated on at least four separate occasions.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Recombinant, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Sonication

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: α 5 β 1 integrin and syndecan-4 regulate p190-A by distinct mechanisms. (A) Phosphotyrosine blots of p190-A immunoprecipitated from high-detergent lysates of fibroblasts prespread on 50K and stimulated with H/0. (B) TrX-soluble p190-A blots of fibroblasts prespread on 50K, inhibited with 10 μM PP2, and stimulated with H/0. (C) Phosphotyrosine blots of p190-A immunoprecipitated from the cytosolic and membrane-bound fractions of fibroblasts prespread on 50K, stimulated with H/0 for 10 min, and lysed by sonication. Loading was adjusted to analyze equal amounts of p190-A from each fraction. Phosphotyrosine blots of p190-A immunoprecipitated from high-detergent lysates of fibroblasts fully spread on fibronectin and 50K (D), spreading on fibronectin (E), spreading on 50K (F), or syndecan-4–null MEFs spreading on fibronectin (G). (H) Cytosolic and membrane-bound p190-A in sonication lysates of fibroblasts spreading on 50K. Equal loading between time points was confirmed by blotting for HSP70 in redistribution experiments and by reprobing blots for p190-A in tyrosine phosphorylation experiments. Error bars represent standard error. *, significant change (P < 0.05) from experiments repeated on at least four separate occasions.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Immunoprecipitation, Sonication

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: Expression of p190-A is necessary for focal adhesion formation in response to syndecan-4 engagement. (A) Wild-type, syndecan-4–null, siRNA-transfected, p190-A–null, or p190-A reexpressing MEFs prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) for 60 min before fixing and staining for vinculin (green) and/or actin (red). Whole cell images are shown in Fig. S2 A (available at http://www.jcb.org/cgi/content/full/jcb.200711129/DC1 ). Bar, 10 μm. (B) Focal adhesion area per cell was quantified digitally, after background subtraction, by measuring the area above an empirically determined threshold of fluorescence intensity within a single experiment. (C) Total area of individual cells. White bars represent cells spread on 50K and black bars represent spread cells after stimulation with H/0. (D and E) Correlation between membrane-bound p190-A (circles), RhoA activity (squares), total focal adhesion area per cell (crosses), and mean focal adhesion length (triangles) in human fibroblasts (D) and MEFs (E). (F) Percentage of human fibroblasts spread on 50K after 2 h in the presence of inhibitory antibodies against α 5 integrin (mAb16), α v integrin (17E6), or nonimmune IgG. Experiments were repeated on four separate occasions. Error bars represent standard error. *, significant change (P < 0.005) from 20 cells per condition.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Expressing, Transfection, Recombinant, Binding Assay, Staining, Fluorescence, Activity Assay

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: p190-A is recruited to lamellae in response to engagement of syndecan-4. Fibroblasts prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) or a heparin-binding mutant (H/0-GAG) before fixation and staining for p190-A (green) and the lipid raft marker GM-1 (red). (A) Immunostaining of p190-A and GM-1, including masks used to measure the pixel intensity of the lamellar border. Bar, 5 μm. (B–E) Pixel intensity of p190-A (B and D) and GM-1 (C and E) within a 0.5-μm-thick border around the edge of each lamella after stimulation with H/0 (B and C) or H/0-GAG (D and E). The pixel intensity of 20 different cells was measured for each time point. The experiment was repeated on six separate occasions. Error bars represent standard error. *, significant change (P < 10 −8 ).

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Recombinant, Binding Assay, Mutagenesis, Staining, Marker, Immunostaining

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: p190-A mediates RhoA regulation in response to engagement of syndecan-4. MEFs prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) before measuring the RhoA activity of lysates by ELISA. Wild-type MEFs (A), p190-A–null MEFs (B), p190-A–null MEFs reexpressing p190-A (C), MEFs transfected with a nontargeting siRNA (E), and MEFs transfected with an siRNA targeted against p190-A (F) are shown. (G) Direct comparison of RhoA activity in cells fully spread on 50K after transfection with a nontargeting or p190-A–targeted siRNA. (D and H) Expression levels of p190-A in null and reexpressing MEFs (D) or MEFs transfected with siRNAs (H); lysates were also probed for the related p190-B isoform to eliminate the possibility of molecular compensation or off-target knockdown. Equal loading between time points was confirmed by a total protein assay. Error bars represent standard error. *, significant change (P < 0.05) from experiments repeated on at least four separate occasions.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Recombinant, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: PKCα mediates syndecan-4–induced redistribution of p190-A. MEFs prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) before preparing lysates and then measuring levels of soluble p190-A in TrX lysates by Western blotting. Wild-type (A) and syndecan-4–null (B) MEFs reexpressing a Y188L mutant syndecan-4 defective in PKCα binding to endogenous levels, treated with 200 nM of the pharmacological PKC inhibitor BIM-1 (C), transfected with a nontargeting siRNA (D), and transfected with an siRNA targeted against PKCα (E). (F) Expression of PKCα after transfection with control or targeting siRNA. (G) Phosphotyrosine blots of p190-A immunoprecipitated from high-detergent lysates of MEFs expressing wild-type or Y188L-syndecan-4 spread on 50K and stimulated with H/0 for 10 min. (H) Phosphorimager scan and blot of p190-A immunoprecipitated from high-detergent lysates of MEFs expressing wild-type or Y188L–syndecan-4, metabolically labeled with [ 32 P]orthophosphate spread on 50K, and stimulated with H/0 for 10 min. RhoA activity ELISAs of MEFs expressing wild-type (I) or Y188L–syndecan-4 (J) prespread on 50K and stimulated with H/0. Equal loading between time points was confirmed by blotting for HSP70 or a total protein assay. Error bars represent standard error. *, significant change (P < 0.05) from experiments repeated on at least five separate occasions.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: Recombinant, Binding Assay, Western Blot, Mutagenesis, Transfection, Expressing, Immunoprecipitation, Metabolic Labelling, Labeling, Activity Assay

Journal: The Journal of Cell Biology

Article Title: p190RhoGAP is the convergence point of adhesion signals from α 5 β 1 integrin and syndecan-4

doi: 10.1083/jcb.200711129

Figure Lengend Snippet: The network of signals arising from transmembrane receptor engagement converge on p190-A. (A) Summary of the connections from α 5 β 1 integrin and syndecan-4 during the initiation of focal adhesion formation. (B) The engagement of receptors, including integrins and cadherins, results in tyrosine phosphorylation of p190-A (gray box). The primed p190-A molecule is itself a target for serine and threonine phosphorylation events, tyrosine dephosphorylation events downstream of syndecan-4 and growth factor receptors, and trafficking molecules such as p120RasGAP. Although there is extensive crosstalk between these pathways, p190-A stands out as a nexus for the multiple signaling events during RhoA regulation.

Article Snippet: Mouse monoclonal antibodies raised against p190-A and phosphotyrosine (4G10; Millipore), vinculin (Sigma-Aldrich), HSP70 (Affinity BioReagents), PKCα (BD Biosciences), and transferrin receptor (Invitrogen); a rat monoclonal antibody against TCP-1α (Assay Designs); and a rabbit polyclonal antibody against p190-B (Cell Signaling Technology) were used according to the manufacturer's instructions.

Techniques: De-Phosphorylation Assay

Journal:

Article Title: The Abl and Arg non-receptor tyrosine kinases regulate different zones of stress fiber, focal adhesion, and contractile network localization in spreading fibroblasts

doi: 10.1002/cm.20479

Figure Lengend Snippet: (A) WT, (B) abl—/—, and (C) arg—/— fibroblasts were plated on 10 μg/mL FN-coated coverslips for one hour, fixed and stained for p120RasGAP, p190RhoGAP, and F-actin. Images were obtained with a 40× objective lens. Merged image (merge) shows p120 (red), p190 (green), and F-actin (blue). NIH ImageJ colocalization image (p120:p190 colocalization) for WT, abl—/—, and arg—/— fibroblasts. Pixels that were above computer-determined threshold values for p120 and p190 are coded white, while other pixels are coded black. Thresholds were set to display the maximal differences in colocalization staining between the cell genotypes. Bar = 10 μm.

Article Snippet: Cells were stained with antibodies to paxillin (Transduction Labs, cat #610051), phospho-Myosin Light Chain 2 (Ser19) (pMLC) (Cell Signaling, cat #3675/3671), p190-A RhoGAP (C59F7 Rabbit mAb Cell Signaling, cat #2860), p120RasGAP (mAb clone B4F8, Upstate), Alexa 594–labeled or Alexa 488-labeled secondary antibodies (Molecular Probes), followed by Alexa 350-phalloidin (Molecular Probes) to visualize the F-actin cytoskeleton.

Techniques: Staining