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: 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