Journal: JCI Insight

Article Title: Targetable purinergic receptors P2Y 12 and A2b antagonistically regulate bladder function

doi: 10.1172/jci.insight.122112

Figure Lengend Snippet: Deletion of P2Y12 and A2b result in enlarged (A) and reduced (B) bladder size, respectively. (C–E) H&E staining images from bladder sections of wild-type, P2Y12-KO, and A2b-KO mice, respectively. (F–H) Immunostaining of Ki67 from bladder sections of wild-type (n = 5), P2Y12-KO (n = 5), and A2b-KO (n = 5) mice, respectively. Positive nuclear staining per bladder section is quantitated in I. (J–L) Integrin β1 immunostaining of bladder sections of wild-type (n = 186 cells), P2Y12-KO (n = 225 cells), and A2b-KO (n = 185 cells) mice, respectively. (M–O) Enlarged images of the white boxes seen in J, K, and L, respectively. (P) Schematic diagram indicating where the bladder was sectioned for staining, and how only bladder smooth muscle (BSM) cells containing nuclei were measured for their cross-section area, which serves as an index of BSM cell size. (Q) BSM cell cross-sectional area was quantitated for each model. (R and S) Quantitative RT-PCR data for c-fos and c-jun mRNA expression in mouse bladder from wild-type (n = 3), P2Y12-KO (n = 3), and A2b-KO (n = 3) mice. (T) Western blot images of c-fos and c-jun protein expression in mouse bladder from wild-type (n = 6), P2Y12-KO (n = 6), and A2b-KO (n = 6) mice, which are quantitated by densitometry and normalized to β-actin in U and V. Data are shown as box and whiskers, whiskers are from minimum to maximum, Student’s t test is used to compare between wild-type and KO animals; *P < 0.05.

Article Snippet: Fixed tissue was cryoprotected, frozen, sectioned, and incubated with rabbit polyclonal anti-Ki67 antibody (catalog ab15580, abcam) and purified rat anti-mouse β1 integrin antibody (catalog 550531, BD Bioscience) (1:100 dilution) overnight at 4°C.

Techniques: Staining, Immunostaining, Quantitative RT-PCR, Expressing, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

doi: 10.1186/ar2024

Figure Lengend Snippet: Over-release of prostaglandin E2 (PGE 2 ) in compressed costal cartilage explants is the result of mechanical stress. (a) Implication of the mechanoreceptor integrin α5β1 in PGE 2 over-release in compressed cartilage explants. Mouse costal cartilage explants treated with either the β1 non-blocking antibody VMA1997 or the α5β1 blocking antibody AB1950 at 2.5 μg/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not-compressed control (cont) value. Data are the mean ± SEM of 2 independent experiments with n = 2/group/experiments, analyzed in duplicate. ***p < 0.001 versus control NC, *p < 0.05 versus control C. (b) Increased PGE 2 release in compressed costal cartilage explants is not due to the cytokine IL-1. Mouse costal cartilage explants treated with the IL-1 receptor antagonist (IL1-Ra) at 100 ng/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not compressed control value. Data are the mean ± SEM of 2 independent experiment with n = 2/group/experiments, analyzed in duplicate. *p < 0.05 versus control NC.

Article Snippet: Anti-goat fibronectin receptor (integrin α5β1) blocking polyclonal antibody (AB1950) was purchased from Euromedex for Chemicon Inc. (Strasbourg, France) and rat anti-mouse β1 subunit of VLA1 integrins non-blocking monoclonal antibody (VMA 1997), was purchased from AbCys SA for Chemicon Inc. (Souffelweyersheim, France).

Techniques: Blocking Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG protein is associated with β1 integrin and activates β1 integrin. a - b . Coimmunoprecipitation. Cell membrane proteins of HCT116 cells were collected and subjected to immunoprecipitated (IP) using anti-SNCG ( a ), anti-SNCG or anti-β1 integrin antibody ( b ). The IP proteins or total cell lysates were analyzed by Western blot. Normal IgG served as the negative control. c . Far-Western blot analysis. HCT116 cells were transfected with control siRNA (lane 1-2), and specific siRNA-β1-2 (lanes 3-4) for 48 h. Cells were treated without (lane 1, 3) or with 1 μmol/L rhSNCG (lane 2, 4). Cell lysates were subjected to SDS-PAGE and transferred to NC membrane. β1 integrin (prey protein) on the membrane is detected with SNCG (bait protein). More SNCG was associated with membrane β1 integrin in SNCG-treated cells than that in the control cells (lane 1, 2). Correspondingly, less SNCG were detected in β1 integrin knock-down cells than that in control cells (lane 2, 4). d - e , Effect of concentration and time treatment of SNCG on activated β1 integrin. HCT116 cells were stimulated with GST or GST-SNCG at various concentrations for 60 min ( d ) or at fixed concentration (1 μmol/L) for various times ( e ). Cell lysates were analyzed with the HUTS-21 mAb recognizing the activated form of β1 integrin. f , GST-SNCG treatment (1 μmol/L) upregulated activated β1 integrin subunit in HCT116 and SW480 cells. g , Colocalization of SNCG with F-actin. HCT116 cells grown on coverslips were transiently transfected with control siRNA or β1-specific siRNA-2. After 72 h, cells were treated with GST or GST-SNCG (1 μmol/L) for 60 min. Cells were fixed and stained with anti-SNCG (red) and FITC-Phalloidin (green). Colocalization of SNCG and F-actin was shown in yellow. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Far Western Blot, Transfection, SDS Page, Concentration Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Integrin β1 is required for enhancement of SNCG on tumor cell migration and invasion. a - b , The functional blocking antibody for β1 integrin subunit (5, 10, 20 μg/mL) was added in the upper compartment of migration or invasion chambers stimulated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( a ) or 48 h for invasion ( b ). c - d , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. then cells were treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( c ) or 48 h for invasion ( d ). e - f , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min and then treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( e ) or 48 h for invasion ( f ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. g , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot. h , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Functional Assay, Blocking Assay, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: FAK is essential for SNCG-enhanced tumor cell migration and invasion. a - b , HCT116 cells were transfected with control siRNA and FAK-specific siRNA-2, -3 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for migration ( a ) or invasion ( b ). c - d , HCT116 cells were treated with PBS, 50 μmol/L FAK inhibitor 14 for 30 min and then treated with or without GST-SNCG (1 μmol/L) for migration ( c ) or invasion ( d ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. e - f , HCT116 cells were transfected with control siRNA (lane 1-2), and FAK-specific siRNA-2 (lane 3-4) and -3 (lane 5-6) for 72 h. cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed for activated and total β1 integrin ( e ) or activated and total FAK ( f )

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG is an indicator of adverse prognosis and positively correlates with activated β1 integrin, p-FAK (Y 397 ) in CRC tissues. a - b , Kaplan-Meier estimation of disease-free survival (DFS) for stage I-II ( a ) and III-IV ( b ) colorectal adenocarcinoma patients according to SNCG levels. c , Correlations of SNCG levels in CRC tissues with post-operative recurrence and status. d , Representative blots from three independent experiments were presented. Protein levels of SNCG, activated β1 integrin, and p-FAK (Y 397 ) in clinical colon cancer tissue samples were evaluated by Western blot analysis. In order to increase the reproducibility, HCT116 cell lysates were used in each blot as the internal control ( c ) to minimize the effect of band intensity variation. GAPDH was used as the loading control. e - g , Correlation between the relative protein levels of activated β1 integrin levels and p-FAK (Y 397 ) ( e ), SNCG and active β1 integrin ( f ), and SNCG and p-FAK (Y 397 ) ( g ) were plotted as a scatter plots

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Exogenously added SNCG remodels the microenvironment of tumor cells and increases MMP-2 activity by β1 integrin. a , Antibody array screening of CM from GST and GST-SNCG-treated HCT116 cells. b , CM (left panel) and whole cell lysates (right panel) from HCT116 and SW480 cells treated with or without GST-SNCG (1 μmol/L) were subjected to Western blot analysis. Representative blots from three independent experiments were presented. c - d , HCT116 cells were treated with diluent, 50 μmol/L MMP-2 inhibitor for 40 min, then 1 μmol/L GST or GST-SNCG was added in the cell medium for migration ( c ) or invasion ( d ) assay. Migrated or invaded cells were quantitated after 24 h or 48 h, respectively. Error bars, SE of three determinations. e - f , Western blot and gelatin zymography analysis. CM from HCT116 cells treated with GST or GST-SNCG (1 μmol/L) in β1 integrin knock-down ( e ) or RGD-treated cells ( f ) was analyzed by gelatin zymography with FBS as the positive control, and secreted protein levels were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Activity Assay, Ab Array, Western Blot, Migration, Zymography, Positive Control

Journal: Molecular Cancer

Article Title: Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

doi: 10.1186/1476-4598-9-126

Figure Lengend Snippet: Arecoline induces detachment of HA22T/VGH cells, followed by apoptosis . (A) HA22T/VGH cells were treated with 0 (left), 30 (center), or 100 (right) μg/ml of arecoline for 24 h or 48 h, then cell morphology was observed under a phase-contrast microscopy at 200× magnification. The black arrows indicate detached cells. (B) After treatment of arecoline for 24 h, β1-integrin expression was measured by flow cytometric analysis using RPE-conjugated antibody. The histogram of red filled area is the untreated control and the black lines the treated groups. The values shown are the mean fluorescence intensity as a percentage of the untreated control value. (C) HA22T/VGH cells or primary normal rat hepatocytes were treated with the indicated concentration of arecoline for 72 h, then viable cells were counted using Trypan blue and the results expressed as a percentage of the untreated control value. (D) After treatment of arecoline for 72 h, the cells were harvested all together or the detached and adherent cells separately and genomic DNA extracted and analyzed on an agarose gel for DNA fragmentation. The left panel shows the total cells and the right panel adherent and detached cells separately. (E) TUNEL staining of the detached cells detected by flow cytometric analysis. The green filled area is the untreated control and the black lines the treated groups. The values shown are the percentage of TUNEL-positive cells in the detached cells. All data are the mean ± S.D. for three independent experiments. *: p < 0.05 as compared to the untreated control.

Article Snippet: R-phycoerythrin (RPE)-conjugated rabbit anti-active caspase-3 polyclonal antibodies, RPE-conjugated mouse anti-human β1-integrin monoclonal antibody and the RPE-conjugated mouse IgG isotype control were purchased from BD Pharmingen Inc. (San Diego, CA, USA).

Techniques: Microscopy, Expressing, Fluorescence, Concentration Assay, Agarose Gel Electrophoresis, TUNEL Assay, Staining