Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits EC migration but not proliferation. ( A ) Representative images from a wound healing scratch assay using control, wild-type TSP1, and phosphomutant TSP1-transfected BPAECs. Scratches were made in a confluent EC monolayer 24 h post-transfection. Images were captured at 0, 8, and 24 h post-scratch. Scale bar = 100 µm. ( B ) Wound closure was evaluated by measuring the open area at each time point, normalized to the 0 h image (**** p < 0.0001). Statistical analysis was completed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD ( n = 5). ( C ) Representative measurement of an in vitro wound healing assay performed using ECIS. Wounding was applied at 1 h. Each line represents the mean of three replicates ± SD. ( D ) Statistical analysis of EC migration rate was performed using one-way ANOVA with Tukey’s test (**** p < 0.0001). Data are represented as mean ± S.D ( n = 8). ( E ) BPAEC were either untransfected (ctr) or transfected with TSP1 WT -, TSP1 S93A -, or TSP1 S93D -expressing constructs. Cell proliferation was measured by an MTT assay. Absorbance values were normalized to 0 h. No significant differences in proliferation were observed between groups. Data represent SD ( n = 6).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Wound Healing Assay, Control, Transfection, In Vitro, Expressing, Construct, MTT Assay

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits FAK signaling and downstream targets during cell migration. ( A ) Control or TSP1-transfected confluent monolayers of BPAECs were scratched 24 h post-transfection. Samples were collected at the indicated time points (0, 4, and 8 h). Overexpression of TSP1 and the levels of signaling protein were analyzed by Western blot. ( B ) Quantitative analysis was performed by densitometry of the Western blot bands. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 3–5) (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Control, Transfection, Over Expression, Western Blot

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D shows enhanced binding to ITGB1. ( A ) Bacterially expressed GST and GST-TSP1 1–221 WT, GST-TSP1 1–221 S93A, and GST-TSP1 1–221 S93D recombinant proteins immobilized on glutathione Sepharose beads were incubated with BPAEC lysate for pull-down assays. EC lysates and the eluted proteins were analyzed by Western blot using ITGB1- and TSP1-specific antibodies. ( B ) Quantitative analysis of pull-down samples. Statistical analysis was performed using one-way ANOVA ( n = 4) (** p < 0.01). ( C ) Control and TSP1-transfected BPAECs were subjected to immunoprecipitation using c-myc antibody to purify recombinant TSP1 proteins. Total cell lysates and immunocomplexes were tested for c-myc and ITGB1 by Western blot. ( D ) Quantitative analysis of IP. Statistical analysis was performed using one-way ANOVA ( n = 4) (*** p < 0.001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Binding Assay, Recombinant, Incubation, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D alters ITGB1 clusterization. ( A ) Representative images of control cells and cells expressing TSP1 recombinant variants, immunostained for ITGB1 (green) and nuclei (DAPI, blue). Images were acquired using the Opera Phenix HCS (PerkinElmer, Inc., Shelton, CT, USA. Scale bar: 100 μm. ( B ) Quantitative analysis of ITGB1 clusters was performed using the built-in Harmony software (version 4.8, Perkin Elmer). Statistical analysis was performed using one-way ANOVA. Data are presented as means ± S.D, and >10,000 cells were analyzed per condition. (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Control, Expressing, Recombinant, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: The S93A mutation of TSP1 increases its secretion via N-glycosylation. ( A ) Cell culture supernatants were analyzed for TSP1 levels using ELISA. Statistical analysis of TSP1 concentration was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 7). Data are reported as means ± S.D. ( B ) TSP1 glycosylation was assessed by immunoprecipitating c-myc–tagged TSP1 proteins from the supernatant of transfected cells. Total cell lysates, supernatants, and IP complexes were tested for c-myc. IP complexes were further analyzed for glycosylation using lectin and O-GlcNac antibody by Western blot. ( C ) Quantification of glycosylation of immunoprecipitated recombinant TSP1 proteins. O-GlcNAc levels were not significantly different among groups. Data are shown as normalized signal intensity. (** p < 0.01 and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Mutagenesis, Glycoproteomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Western Blot, Immunoprecipitation, Recombinant

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D enhances SMC migration and proliferation and induces morphological changes. ( A ) MOVAS cells were seeded at 10% confluence and treated with conditioned media from non-transfected (ctr) or TSP1-transfected BPAECs. Proliferation was assessed using MTT, with absorbance measured at 540 nm at 24, 48, and 72 h. Data are shown as means ± SEM ( n = 12). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.01). ( B ) Migration of treated MOVAS cells was measured using an ECIS-based wound healing assay. Data presented mean ± S.D. from three chambers per condition. ( C ) Statistical analysis of migration rates was performed using one-way ANOVA with Tukey’s post hoc test ( n = 5; means ± S.D.; **** p < 0.0001). ( D ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. White arrows indicate filopodia of cells. Scale bars: 50 μm. ( E ) Morphological parameters of MOVAS cells were analyzed using Harmony software on the Opera Phenix HCS system. Data are presented as mean ± SD (1000–1600 cells per well, n = 4). Statistical analysis was performed using ANOVA (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Transfection, Wound Healing Assay, Control, Staining, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D induces a shift toward a synthetic-like state in SMCs and increases IL-6 secretion. ( A ) MOVAS were treated with conditioned media from TSP1 overexpressing ECs. Expression levels of indicated proteins were tested by Western blot. Actin was used as a loading control. ( B ) Densitometric analysis of Western blot signals. Statistical analysis was performed using one-way ANOVA with Tukey’s test ( n = 4). ( C ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. Scale bars: 50 μm. ( D ) Conditioned media treated and untreated MOVAS cell supernatants were analyzed by ELISA for IL-6. Statistical analysis was performed using one-way ANOVA ( n = 9). Data are reported as means ± S.D. (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Expressing, Western Blot, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: Expression of THBS1 in paired tumor and adjacent normal tissues, and correlation analysis between FGFR2 and THBS1 expression. (A–D) Representative micrographs showing higher THBS1 staining and lower THBS1 staining in human gastric cancer tissue (A and B) and adjacent normal tissues (C and D) (magnification, ×200). (E) Quantification of THBS1 expression in tumor tissues compared with adjacent normal tissues. Data are presented as the mean ± SD ( ** P<0.01). (F) Correlation analysis of FGFR2 and THBS1 expression.

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing, Staining

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: Correlation of THBS1 expression with clinicopathological factors.

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: FGF7 elevates the expression of THBS1 in vitro . (A–D) Cells were incubated with FGF7 (10 ng/ml) for the indicated times. The expression of FGFR2, p-FGFR, THBS1 was detected by western blotting (A). The relative expression of THBS1 mRNA was detected by qRT-PCR (C). FGFR2 and THBS1 expression (B) and relative expression of THBS1 mRNA (D) were detected after treatment with FGF7 (10 ng/ml for 48 h) in shRNA-2 and shRNA-NC transfected cells and SGC7901 cells. (E) THBS1 secreted by cells treated with or without FGF7 for 12, 24 and 48 h was determined by ELISA. (F) THBS1 in the condition media of shRNA-2 and shRNA-NC transfected cells treated with or without FGF7 was detected by ELISA. Data are presented as the mean ± SD of three independent experiments ( * P<0.05, ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing, In Vitro, Incubation, Western Blot, Quantitative RT-PCR, shRNA, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: THBS1 is required for the effect of FGF7 on invasion and migration in gastric cancer cells. (A) Cancer cell lines were treated with or without FGF7, THBS1 in the condition media were detected by ELISA. Western blotting (B) and qRT-PCR (C) were used to evaluate the efficiency of THBS1 knockdown in SGC7901 cells transfected with siRNA-1, siRNA-2, siRNA-3 and siRNA-NC. (D and E) Cells transfected with siRNA-2 and siRNA-NC were treated with FGF7 (10 ng/ml) and subjected to invasion and migration assays. Data are presented as the mean ± SD of three independent experiments ( ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Knockdown, Transfection

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: FGF7 regulates THBS1 through PI3K/Akt/mTOR pathway in vitro . (A and B) Cells were pretreated with FGF7 (10 ng/ml for 48 h), and THBS1 was assessed by performing western blotting after treated with indicated concentration of LY294002, U0126, SB203580, SP600125 (A) and RAD001 (B) at different time-points. (C) Relative expression of THBS1 mRNA in cells treated with RAD001 (pretreated with FGF7) was assessed by qRT-PCR. Data are presented as the mean ± SD of three independent experiments ( ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: In Vitro, Western Blot, Concentration Assay, Expressing, Quantitative RT-PCR