Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deficiency of Micro RNA miR ‐1954 Promotes Cardiac Remodeling and Fibrosis

doi: 10.1161/JAHA.119.012880

Figure Lengend Snippet: Overexpression of miR‐1954 reduces THBS 1 (thrombospondin 1) in mouse model with angiotensin II (Ang II ) infusion in vivo. A , Alignment between THBS 1 and miR‐1954 as predicted by TargetScan 7.1 and miRbase analyses, as shown by vertical bars. The sequence alignment of putative miR‐1954 and its targeting site on 3′ untranslated region of THBS 1 shows a high level of complementarity. B , Determination of THBS 1 expression and level in Ang II ‐treated wild‐type ( WT) and miR‐1954 mice. The mRNA expression of THBS 1 was determined by quantitative reverse transcription polymerase chain reaction (5–8 per group). GAPDH was used as an internal control. C , THBS 1 protein level was determined by THBS 1‐specific antibody (3–6 per group). GAPDH was used as an internal protein loading control. D , Quantification of Western blotting. Data are expressed as mean± SE . * P <0.0001 vs WT ; $ P <0.0001 vs WT +AngII.

Article Snippet: The miRNA 3′ untranslated region (UTR) target expression clone for mouse THBS1 (NM_001313914.1) in pEZX‐FR02 and the corresponding control vector firefly luciferase and Renilla luciferase were made from GeneCopoeia.

Techniques: Over Expression, In Vivo, Sequencing, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deficiency of Micro RNA miR ‐1954 Promotes Cardiac Remodeling and Fibrosis

doi: 10.1161/JAHA.119.012880

Figure Lengend Snippet: Angiotensin II (Ang II ) stimulation downregulates miR‐1954 expression and upregulates THBS 1 (thrombospondin 1) expression in cardiac fibroblasts. A , Determination of miR‐1954 expression in cardiac myocytes ( CM ) and cardiac fibroblast ( CF ). B , CFs were treated with 1 µmol/L Ang II for 48 hours. Mature miR‐1954 expression was determined by quantitative reverse transcription polymerase chain reaction ( qRT ‐ PCR ). U6 was used as an internal control. C , Gene expression of THBS 1 is a likely a target for miR‐1954. CFs were transfected with scramble sequence, miR‐1954 mimic, and miR‐1954 inhibitor for 24 hours, followed by Ang II treatment for another 48 hours. Gene expression of THBS 1 was determined by qRT ‐ PCR . GAPDH was used as an internal control. Data are expressed as mean± SE from 3 to 5 independent experiments. * P <0.0001 vs control; & P <0.001 vs mimic+Ang II ; $ P <0.05 vs inhibitor. Student t test was used in panel B; 1‐way ANOVA with Tukey post test was used in panel C. D , Neonatal CFs were transfected with an empty vector, THBS 1 3′ untranslated region ( UTR) luciferase construct, THBS 1 3′ UTR luciferase construct with miR‐1954 mimic (miR‐1954 M) and THBS 1 3′ UTR luciferase construct with miR‐1954 inhibitor (miR‐1954 I). After 48 hours of transfection, cells were collected, and firefly luciferase activities were estimated and normalized to Renilla luciferase activity. Data are expressed as mean± SE from 3 to 5 independent experiments. P values for comparisons of columns are demonstrated in the figure.

Article Snippet: The miRNA 3′ untranslated region (UTR) target expression clone for mouse THBS1 (NM_001313914.1) in pEZX‐FR02 and the corresponding control vector firefly luciferase and Renilla luciferase were made from GeneCopoeia.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Gene Expression, Transfection, Sequencing, Plasmid Preparation, Luciferase, Construct, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of the TGF-β1/thrombospondin-1 axis.

doi: 10.1016/j.matbio.2022.03.003

Figure Lengend Snippet: Figure 3. THBS1 is the key ECM protein for adhesion whose expression is downregulated by vitamin D. (A) Schematic protocol for gene expression analysis in TGF-b1 (10 ng/ml) -stimulated MCs treated with vehicle (DMSO) or vitamin D (10 mM) for 72 h. (B) Heat map depicting 549 differentially expressed genes (change > two-fold, P < 0.05) in TGF-b1-stimulated MCs treated with or without vitamin D. (C) Principal component analysis (PCA) map illus- trating similarities in gene expression between each sample group. (D) Gene Set Enrichment Analysis (GSEA) in TGF- b1-stimulated MCs without vitamin D compared to those cultured with vitamin D. NES: Normalized enrichment score. (E) Pathway analysis was performed with significantly differentially expressed genes in MCs stimulated by TGF-b1 with or without vitamin D. (F) Gene Ontology (GO) analysis was performed with proteins that were coded by significantly differen- tially expressed genes in MCs stimulated by TGF-b1 with or without vitamin D. (G) Volcano plot of 7,864 identified genes encoding proteins. The colored dots show each pathway related genes, which were significantly differentially expressed; green dots show focal adhesion related genes, purple dots show TGF-b pathway related genes, and orange dots show both pathway related genes. (H) List of 13 genes whose differential expression changed significantly and were involved in either the focal adhesion or TGF-b pathway, indicating the specific fold-change and P -value for each gene. (I) Interac- tome analysis was performed using the 13 genes shown in Figure 3H showing significant changes on expression levels in MCs stimulated by TGF-b1 with or without vitamin D. MCs: mesothelial cells, VD: vitamin D, EMT: epithelial-mesenchy- mal transition, THBS1: thrombospondin-1.

Article Snippet: For analyzing cell adhesion following THBS1 treatment, confluent monolayers of MCs were incubated with or without recombinant human THBS1 (0.1 nM, 1 nM, and 10 nM; catalog PKSH033500, Elabscience Biotechnology Inc., TX, USA) for 5 h, after which, the supernatant was removed and the cells were washed twice with serum-free medium.

Techniques: Expressing, Gene Expression, Cell Culture, Quantitative Proteomics

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of the TGF-β1/thrombospondin-1 axis.

doi: 10.1016/j.matbio.2022.03.003

Figure Lengend Snippet: Figure 4. Vitamin D inhibited adhesion and proliferation of OvCa cells by suppressing of THBS1 via VDR/Smad3 competition. (A) Schematic showing the protocol used for investigating the effect of vitamin D addition on TGF-b1-stimulation to MCs. (B) Representative immunofluorescence of MCs treated with vehicle (DMSO; Ctrl), TGF-b1 (10 ng/ml) þ vehicle, and TGF-b1 þ vitamin D (10 mM) for 72 h. Immunofluorescence for THBS1 (magenta) and calretinin (green) with nuclear DAPI staining (blue). Scale bars, 50 mm. n = 3. (C and D) TGF-b1 increases THBS1 expression, which is alleviated by vitamin D (10 mM). Results of representative western blotting and real-time PCR. n = 3. (E) Representative images of GFP-labeled ES-2 cells adhering to treated MCs. Scale bars, 100 mm. (F) Results of luciferase activity measurements representing cancer cells adhering to treated MCs. n = 3. (G) Representative images of GFP-labeled ES-2 cells, which proliferated on treated MCs. Scale bars, 100 mm. n = 3. (H) Results of luciferase activity measurements representing can- cer cells which proliferated on treated MCs. n = 3. (I) Schematic protocol to investigate the effect of vitamin D addition on TGF-b1-stimulation of human omental tissues without malignant tumor. (J) Representative images of GFP-labeled ES-2 cells adhering to omental tissues treated as in Figure 4I. Scale bars, 500 mm. n = 3. (K) Results of luciferase activity meas- urements representing cancer cells adhering to treated omentum. (L) Schematic protocol showing siRNA suppression to investigate the effect of THBS1 in the interaction between CAMs and cancer cells. (M) Relative expression levels of THBS1 mRNA in CAMs after siRNA treatment and representative western blot showing THBS1 expression. n = 3. (N) Representative images of GFP-labeled ES-2 cells adhering to treated CAMs. Scale bars, 100 mm. n = 3. Luciferase

Article Snippet: For analyzing cell adhesion following THBS1 treatment, confluent monolayers of MCs were incubated with or without recombinant human THBS1 (0.1 nM, 1 nM, and 10 nM; catalog PKSH033500, Elabscience Biotechnology Inc., TX, USA) for 5 h, after which, the supernatant was removed and the cells were washed twice with serum-free medium.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Labeling, Luciferase, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of the TGF-β1/thrombospondin-1 axis.

doi: 10.1016/j.matbio.2022.03.003

Figure Lengend Snippet: Figure 5. Vitamin D restored TGF-b1-induced EMT and THBS1 expression in MCs. (A) Omental metastasis tumoroids were established from OvCa tissues. (B) Phase-contrast images of tumoroids at 5 and 15 days after culture initiation. The tumoroids were treated with vehicle (DMSO; Ctrl) or vitamin D (10 mM) for 10 d. T: tumor cells, scale bars, 50 mm. Representative immunofluorescence on day 5 for EpCAM (magenta) and calretinin(green) along with nuclear DAPI staining (blue) without addition of vitamin D. Scale bars, 50 mm. n = 3. (C) Representative immu- nofluorescence for calretinin (green), E-cadherin (magenta), F-actin (green), and THBS1 (magenta) with nuclear DAPI staining (blue) on day 15 with addition of vehicle (DMSO; Ctrl) or vitamin D (10 mM). Scale bars, 50 mm. n = 3. (D) Meas- urements of the aspect ratio (longest axis/perpendicular axis). n = 10. (E) Flow cytometry schematic protocol for sorting

Article Snippet: For analyzing cell adhesion following THBS1 treatment, confluent monolayers of MCs were incubated with or without recombinant human THBS1 (0.1 nM, 1 nM, and 10 nM; catalog PKSH033500, Elabscience Biotechnology Inc., TX, USA) for 5 h, after which, the supernatant was removed and the cells were washed twice with serum-free medium.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of the TGF-β1/thrombospondin-1 axis.

doi: 10.1016/j.matbio.2022.03.003

Figure Lengend Snippet: Figure 6. Vitamin D protected peritoneal microvilli and decreased peritoneal dissemination in vivo. (A) Schematic protocol used for in vivo mouse experiments for investigating the peritoneal protective effect of vitamin D. (B and C) Western blot analysis and immunofluorescence for THBS1 (magenta) and F-actin (green) expression on the mice peritoneal surface treated with vehicle (DMSO: Ctrl), TGF-b1 þ vehicle or TGF-b1 þ vitamin D. Scale bars, 100 mm. (D) Representative SEM images of the peritoneal surface treated with vehicle (DMSO; Ctrl), TGF-b1 þ vehicle or TGF- b1 þ vitamin D. n = 6. Scale bars, 5 mm. (E) Quantitative analysis of the damaged microvilli areas of SEM images using the ImageJ software. n = 4. (F) Schematic protocol to confirm the reduction of OvCa cell adhesion to the peritoneum in response to vitamin D treatment. (G) Representative images from fluorescence stereoscopic microscope of the mesente- rium and omentum treated with TGF-b1 þ vehicle or TGF-b1 þ vitamin D. Scale bars, 1 mm. (H) The number of meta- static tumors (3 0.2 mm) were determined 2 d after injection. n = 3. (I) Peritoneal dissemination to the mesenterium was evaluated using stereoscopic microscopy images using the ImageJ software. n = 4. (J) Schematic protocol to confirm the peritoneal restoration effect of vitamin D. (K) Western blot analysis and immunofluorescence showing THBS1 expression on the mice peritoneal surface treated with vehicle (DMSO; Ctrl) or vitamin D after TGF-b1 stimulation. Scale bars, 100 mm. (L) Model of the VDR suppression mechanism of THBS1 transcription in MCs. (M) Hypothetical model describing the effect of vitamin D for plasticity of MCs in the tumor microenvironment during peritoneal dissemination of OvCa. *P < 0.05, ***P < 0.001, ****P < 0.0001, unpaired, two-tailed Student’s t-test. Error bars in all data indicate mean§SEM. MCs: mesothelial cells, VD: vitamin D, VDR: vitamin D receptor, THBS1: thrombospondin-1, EMT: epithelial-mesenchymal tran- sition, MET: mesenchymal-epithelial transition.

Article Snippet: For analyzing cell adhesion following THBS1 treatment, confluent monolayers of MCs were incubated with or without recombinant human THBS1 (0.1 nM, 1 nM, and 10 nM; catalog PKSH033500, Elabscience Biotechnology Inc., TX, USA) for 5 h, after which, the supernatant was removed and the cells were washed twice with serum-free medium.

Techniques: In Vivo, Western Blot, Immunofluorescence, Expressing, Software, Fluorescence, Microscopy, Injection, Two Tailed Test

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: Cell Cycle

Article Title: Thrombospondin-1 counteracts the p97 inhibitor CB-5083 in colon carcinoma cells

doi: 10.1080/15384101.2020.1754584

Figure Lengend Snippet: The steady-state level of THBS1 protein is increased by CB-5083. (A) HCT116 cells were treated with the indicated concentrations of CB-5083, Bortezomib and MLN4924 for 8 h. The steady-state levels of THBS1 and indicated proteins were analyzed by immunoblotting. Actin was used as a loading control. (B) Relative abundance of THBS1 protein was quantified (N = 3, SD error). (C) Relative abundance of known biomarkers of p97 inhibition (HERP1, CHOP and ATF4) was quantified (N = 3, SD error). (D) Mean fold changes (N = 3, SD error) for CDT1 and p21 with 3 μM CB-5083, 1 μM Bortezomib and 3 μM MLN4924. (E) An immunofluorescence assay for THBS1 (green), Actin (red) and DAPI (blue) in HCT116 cells treated with or without 1.5 μM CB-5083 for 8 h (N = 3, SD error).

Article Snippet: Overexpression and knockdown of THBS1 Human THBS1 cloned into pCMV3-Flag-THBS1 plasmid was purchased from Sino Biological (catalog number HG10508-NF).

Techniques: Western Blot, Control, Inhibition, Immunofluorescence

Journal: Cell Cycle

Article Title: Thrombospondin-1 counteracts the p97 inhibitor CB-5083 in colon carcinoma cells

doi: 10.1080/15384101.2020.1754584

Figure Lengend Snippet: Knockdown of THBS1 sensitizes cells to CB-5083 and promotes apoptosis. (A) HCT116 cells transfected with siControl or siTHBS1-1 (shortly siTHBS1) were treated with or without 0.75 μM CB-5083 for 8 h. Total extracts were analyzed by immunoblotting. Actin was used as a loading control. (B) HCT116 cells transfected with siControl or siTHBS1 were treated with or without 0.375 μM CB-5083 for 72 h. Cells stained with crystal violet and are shown. (N = 3, SD error). *p < 0.05. (C) HCT116 cells transfected with siControl or siTHBS1 were treated with or without 0.375 μM CB-5083. Cell numbers were counted at 24 h intervals (N = 3, SD error). *p < 0.05. (D) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083. Cell viability was analyzed by quantifying cellular ATP levels (N = 3, SD error). *p < 0.05. (E) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083 for 72 h. Cell cycle was analyzed by flow cytometry and the percent Sub-G1 is shown (N = 3, SD error). *p < 0.05. (F) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083 for 72 h. Caspase 3/7 activities were measured by immunofluorescence (N = 3, SD error). *p < 0.05.

Article Snippet: Overexpression and knockdown of THBS1 Human THBS1 cloned into pCMV3-Flag-THBS1 plasmid was purchased from Sino Biological (catalog number HG10508-NF).

Techniques: Knockdown, Transfection, Western Blot, Control, Staining, Flow Cytometry, Immunofluorescence

Journal: Scientific Reports

Article Title: Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells

doi: 10.1038/s41598-019-51152-6

Figure Lengend Snippet: IFNT induces cell survival and counteracts THBS1 apoptotic actions. LGCs were treated with either with basal media (control), roIFNT (1 ng/mL), human recombinant THBS1 (250 ng/mL), or the combination of IFNT and THBS1 for 48 h. ( a ) The viable cell number, ( b ) Representative images of the western blots for each antibody, ( c ) XIAP and ( d ) cleaved caspase-3 proteins. Protein levels were determined in cell extracts by western blotting using specific antibodies and normalized relative to the abundance of total MAPK (p44/42). The results represent means ± SEM of four independent experiments. Asterisks indicate significant differences from their respective controls (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Then cells were incubated for the times indicated in the legends either with basal media (media containing 1% FCS) or with roIFNT (0.01–10 ng/mL; a generous gift from Prof. Fuller W. Bazer, Texas A&M University) or recombinant human THBS1 (rhTHBS1; 250 ng/mL; Genentech, South San Francisco, CA) alone or with the combination of rhTHBS1 and roIFNT.

Techniques: Recombinant, Western Blot

Journal: Scientific Reports

Article Title: Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells

doi: 10.1038/s41598-019-51152-6

Figure Lengend Snippet: List of primers used for qRT-PCR.

Article Snippet: Then cells were incubated for the times indicated in the legends either with basal media (media containing 1% FCS) or with roIFNT (0.01–10 ng/mL; a generous gift from Prof. Fuller W. Bazer, Texas A&M University) or recombinant human THBS1 (rhTHBS1; 250 ng/mL; Genentech, South San Francisco, CA) alone or with the combination of rhTHBS1 and roIFNT.

Techniques: Sequencing