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: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 2. Thrombospondin-1 (THBS1) is necessary and sufficient for the morphological ‘transformation’ of wildtype (WT) organoids. (A) Quantification of the percentage of WT cystic organoids in T-cM upon neutralisation with blocking antibodies against ceruloplasmin (CP), connective tissue growth factor (CTGF), hepatoma-derived growth factor (HDGF), galectin-3 (LGALS3), galectin-3 binding protein (LGALS3BP), thrombospondin-1 (THBS1), and transthyretin (TTR) (5 µg/ml). (B) Quantification of the percentage of WT cystic organoids in T-cM upon neutralisation with three different blocking

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Transformation Assay, Blocking Assay, Derivative Assay, Binding Assay

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 3. Thrombospondin-1 (THBS1) is essential for the growth of tumoroids. (A–D) Representative bright-field images of wildtype (WT) organoids (A, B) or tumoroids (C, D) incubated with IgG1 isotype control antibodies (A, C) or anti-THBS1 A6.1-neutralising antibody (B, D) (10 µg/ml). (E, F) Representative images of tumoroids infected with a lentivirus CRISPR-GFP without sgRNA (control in E) or with an sgRNA targeting Thbs1 (Thbs1- KO in F) 48 hr after replacement of single-cell seeding medium (ENRC) by tumoroid medium (EN). (G, H) Quantification of the number of tumoroids

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Incubation, Control, Infection, CRISPR

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 5. Thbs1 is expressed by Lgr5+ cancer stem cells in vivo and induces YAP activation in neighbouring epithelial cells. (A, C) Representative section of Apc mutant intestinal tumours analysed by single-molecule fluorescence in situ hybridisation (smFISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in A) or the YAP target Sca1 (pSca1, green dots in C). Examples of segmented and processed region of interest (ROI) that were automatically counted as co-localisation (Thbs1+/Lgr5+ in A or Thbs1+/Sca1+ cells in C outlined in yellow and indicated by yellow arrows) or single-probe expression (outlined in red or green and indicated by arrows of the corresponding colour) are shown. E-cadherin demarcates epithelial cells in white and DAPI labels nuclei in blue in (A) and (C). (B, D) Quantification of the frequency of tumour regions expressing exclusively one probe or co-expressing two probes (yellow): Thbs1 only in red or Lgr5 only in green (B); Thbs1 only in red or Sca1 only in green (D). The observed frequencies of co-localisation (yellow in B) or mutual exclusion (yellow in D) are statistically significant compared to the calculated probability of random co- expression (blue columns) (n = 22 sections from two tumours in B and n = 51 sections from five tumours in D). (E) Correlation of the number of RNA molecules (dots/mm²) detected by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for the YAP target CTGF and Thbs1 in mouse intestinal tumours. Red dots indicate large tumours (≥ 8 mm), orange dots small tumours (<8 mm). Dashed lines indicate 95% confidence intervals. (F, G) Representative sections of tumours derived from VillinCreERT2;Apcflox/+ (Apc+/- in F) or VillinCreERT2;Apcflox/flox (Apc-/- in G) immunostained for YAP1 (in red)

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: In Vivo, Activation Assay, Mutagenesis, Fluorescence, In Situ, Hybridization, Expressing, Derivative Assay

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 6. The THBS1-YAP pathway operates in human low-grade adenomas. (A) Correlation matrix between the expression levels of THBS1 and the YAP targets CTGF, CYR61, and LGR5 in human colon tumours from the TCGA colon cancer bulk datasets. R indicates Spearman’s coefficient. (B– E) Representative sections of low-grade human adenomas (B, D) or advanced human carcinomas (C, E) processed by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in B, C) or immunostained with anti-YAP1 antibodies (D, E). White arrows highlight tumour cells presenting high nuclear YAP in (D, E). n = 5 human low-grade adenomas in (B, D) and n = 5 advanced human adenocarcinomas in (C, E). (F) Graphical summary of paracrine interactions between wildtype (WT) organoids and tumoroids along the THBS1-YAP axis. Mutant tumoroids ‘corrupt’ genetically WT organoids by secreting THBS-1 (orange arrows). This results in YAP1 nuclear translocation (black nuclei in organoids or tumoroids) and ectopic proliferation as well as cystic morphology in a subset of organoids.

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Expressing, Fluorescence, In Situ, Hybridization, Mutagenesis, Translocation Assay

Journal: Annals of Hematology

Article Title: Lycorine inhibits the proliferation of acute myeloid leukemia cells by upregulating the expression of THBS1

doi: 10.1007/s00277-026-06777-9

Figure Lengend Snippet: THBS1 is a potential target for the action of lycorine in AML. ( a ) Volcano plots comparing the expression fold changes of differentially expressed genes in AML patients versus the control group. ( b ) Hierarchical clustering heatmaps showing differentially expressed genes. ( c ) Molecular docking of lycorine and THBS1

Article Snippet: The THBS1 ELISA kit was purchased from Proteintech Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Control

Journal: Annals of Hematology

Article Title: Lycorine inhibits the proliferation of acute myeloid leukemia cells by upregulating the expression of THBS1

doi: 10.1007/s00277-026-06777-9

Figure Lengend Snippet: The expression of THBS1 is downregulated in AML, while lycorine is capable of upregulating its expression level. ( a ) Real time PCR detection of THBS1 mRNA levels in bone marrow mononuclear cells of AML patients and control group. ( b ) ELISA detection of THBS1 levels in the serum of AML patients and control group. ( c - d ) Boxplot represents the expression levels of THBS1 in AML and normal tissues derived from the R2 database. ( e - f ) THBS1 expression in HL-60 and THP-1 detected by real time PCR and Western blot. ( g ) THBS1 secretion of HL-60 and THP-1 detected by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: The THBS1 ELISA kit was purchased from Proteintech Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot

Journal: Annals of Hematology

Article Title: Lycorine inhibits the proliferation of acute myeloid leukemia cells by upregulating the expression of THBS1

doi: 10.1007/s00277-026-06777-9

Figure Lengend Snippet: Overexpression of THBS1 inhibits AML cells proliferation and promotes apoptosis. ( a - b ) THBS1 expression in HL-60 and THP-1 detected by real time PCR and Western blot. ( c ) THBS1 secretion of HL-60 and THP-1 detected by ELISA. ( d ) EdU assays evaluated that transfection of THBS1 overexpression plasmid inhibited the proliferation levels of HL-60 and THP-1. ( e ) GSEA-based KEGG-enrichment plots of representative gene sets from activated pathway. ( f ) FCM assays evaluated that transfection of the THBS1 overexpression plasmid promoted the apoptosis levels of HL-60 and THP-1. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The THBS1 ELISA kit was purchased from Proteintech Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/eLife.76541

Figure Lengend Snippet:

Article Snippet: Transfected construct , LentiThbs1-FLAG , Origene , MR211744L3V , .

Techniques: Transduction, Control, Derivative Assay, Cell Recovery, Plasmid Preparation, Multiplex sample analysis, RNAscope, Multiplex Assay, Recombinant, Sequencing, Software, Transfection, Construct

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

Journal: Drug Design, Development and Therapy

Article Title: Tanshinone IIA Ameliorates Streptozotocin-Induced Diabetic Nephropathy, Partly by Attenuating PERK Pathway-Induced Fibrosis

doi: 10.2147/DDDT.S257734

Figure Lengend Snippet: Tan IIA down-regulates TGF-β1, TSP-1, Grp78 and CHOP expression in the renal tissues of the diabetic rats. ( A ) Immunohistochemical analysis of TGF-β1, TSP-1, Grp78 and CHOP expression in the renal tissues among five groups and the pooled data from ten sections for each group is summarized. Scale bar: 50 μm (×400). ( B, C ) The expression levels of Grp78 ( B ) and CHOP ( C ) from different treatment groups were determined by qRT-PCR. N = 10; * P <0.05 and ** P <0.01.

Article Snippet: The sections were incubated with primary antibodies against Grp78 (1:100), CHOP (1:50), TGF-β1 (1:100) and TSP-1 (1:100) overnight at 4°C, followed by incubating with biotinylated goat anti-rabbit IgG (1:200, # BA1003; Boster Biological Technology, Wuhan, China) in phosphate-buffered saline (PBS) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemical staining, Quantitative RT-PCR