Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet: ( a ) Upper panels, total flow cytometry plots of HUVEC and TIME cells stained for endothelial cell markers PECAM and von Willebrand factor (vWF). Medium and lower panels, surface flow cytometry plots of HUVEC and TIME cells stained for PCDH1, β3 integrin, DAF, β1 integrin. ( b ) Surface flow cytometry of wild-type (WT) and knockout (KO) TIME cells stained as above. Histograms of WT cells are shown in gray; single- and double-KO cells are shown in color. ( c ) Western blot analysis of WT TIME cells and KO cells ± cDNA. β-Actin was used as a loading control. Figure 1—source data 1. Original blot of WT TIME cells and KO cells ± cDNA.

Article Snippet: Antibody , APC-α-Human-β1-Integrin (Mouse monoclonal) , BD , Cat. # 59883 , Flow 1:20.

Techniques: Flow Cytometry, Staining, Knock-Out, Western Blot

Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet:

Article Snippet: Antibody , APC-α-Human-β1-Integrin (Mouse monoclonal) , BD , Cat. # 59883 , Flow 1:20.

Techniques: Plasmid Preparation, Expressing, Transduction, Recombinant, Sequencing, Staining, Software, Imaging

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:

Article Title: Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin

doi:

Figure Lengend Snippet: Cleavage of E-cadherin, but not occludin, by BFT. (A) Occludin immunoblot of HT29/C1 cells. HT29/C1 cells were treated with BFT (5 nM) for various times, lysed, and examined by Western blotting using a polyclonal anti-human occludin antibody. Occludin is a 65-kDa protein (arrow). Lane 1, untreated HT29/C1 cells; lanes 2–5, BFT for 30 min, 60 min, 2 hr, 3 hr, respectively; lane 6, untreated HT29/C1 cells. (B) Time course of BFT proteolysis of E-cadherin. HT29/C1 cells were treated for various times with BFT (5 nM), lysed in 1× SDS/gel loading buffer, and examined by Western blot using the E2 antibody (34). Cell-shape changes were observed beginning 10 min after treatment with BFT, with 100% of cells affected by 30 min. Arrows indicate intact E-cadherin (120 kDa) and E-cadherin fragments (33 and 28 kDa) observed after BFT treatment. Lane 1, untreated HT29/C1 cells; lanes 2–10, BFT for 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 1 hr, 2 h, 3 hr, respectively. Immunostaining of the housekeeping protein GAPDH showed no change over time. (C) Concentration dependency of proteolysis of E-cadherin by BFT. HT29/C1 cells were treated with 0.005–5 nM BFT for 30 min. The immunoblot was processed and probed with E2 antibody as in 1B. Lane 1, untreated HT29/C1 cells; lane 2, 0.005 nM BFT; lane 3, 0.05 nM BFT; lane 4, 0.5 nM BFT; lane 5, 5 nM BFT. (D) Time course of BFT proteolysis of E-cadherin expressed by LE cells. LE cells (E-cadherin-transfected L cells) were induced overnight with 1 μM dexamethasone followed by treatment for various times with BFT (5 nM). The immunoblot was processed and probed with the E2 antibody as in Fig. ​Fig.11B. Lane 1, control LE cells; lanes 2–6, BFT for 5 min, 10 min, 15 min, 30 min, 1 hr, respectively. (E) Effect of CCCP on BFT-stimulated E-cadherin proteolysis. HT29/C1 cells were treated with CCCP as described in Materials and Methods. CCCP-treated HT29/C1 cells were compared with HT29/C1 cells treated with BFT (5 nM) in standard HT29/C1 medium. Lanes 1–4, HT29/C1 cells without CCCP treatment. Lane 1, control HT29/C1 cells; lanes 2–4, BFT for 10 min, 30 min, 60 min, respectively. Lanes 5–8, HT29/C1 cells treated with CCCP. Lane 5, BFT for 10 min, 30 min, 60 min, control HT29/C1 cells, respectively.

Article Snippet: HT29/C1 cells were treated with BFT (5 nM) for 30 or 60 min followed by fixation with 10% formaldehyde, immunostaining with either a rat IgG mAb to the extracellular domain of E-cadherin (Decma1 antibody, Sigma) or a polyclonal mouse anti-human β 1 -integrin IgG antibody (Upstate Biotechnology, Lake Placid, NY).

Techniques: Western Blot, SDS-Gel, Immunostaining, Concentration Assay, Transfection

Journal:

Article Title: Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin

doi:

Figure Lengend Snippet: Immunofluorescent confocal microscopy of BFT-treated HT29/C1 cells. (A) Control β1-integrin. (B) Control E-cadherin. (C) Control Nomarski optics. Edges of individual control (untreated) cells are indistinct by Nomarski optics. (D) Thirty minutes after BFT-β1-integrin treatment. (E) Thirty minutes after BFT-E-cadherin treatment. (F) Thirty-minute BFT-Nomarski optics. Note the dramatic loss of E-cadherin immunofluorescence without a change in β1-integrin immunofluorescence. By Nomarski optics, HT29/C1 cellular borders are more distinct in areas of loss of E-cadherin immunofluorescence but remain indistinct where E-cadherin immunofluorescence remains intact. (G) Sixty minutes after BFT- β1-integrin treatment. (H) Sixty minutes after BFT-E-cadherin treatment. (I) Sixty-minute BFT-Nomarski optics. By Nomarski optics, individual HT29/C1 cell borders are visible. (Magnification, ×1,200.)

Article Snippet: HT29/C1 cells were treated with BFT (5 nM) for 30 or 60 min followed by fixation with 10% formaldehyde, immunostaining with either a rat IgG mAb to the extracellular domain of E-cadherin (Decma1 antibody, Sigma) or a polyclonal mouse anti-human β 1 -integrin IgG antibody (Upstate Biotechnology, Lake Placid, NY).

Techniques: Confocal Microscopy, Immunofluorescence

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