Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: EMT‐related protein and marker expression induced by negative‐pressure in vitro. (a) Enzyme‐linked immunosorbent assay (ELISA) of culture supernatants and cell lysates revealed significantly increased protein levels of thrombospondin‐1 (THBS1) ( p = 0.008), transforming growth factor‐beta 1 (TGF‐β1) ( p = 0.008), plasminogen activator inhibitor‐1 (PAI‐1) ( p = 0.001) and hypoxia‐inducible factor‐1 alpha (HIF‐1α) ( p = 0.022) in keratinocytes cultured under negative‐pressure (NP) compared with ambient pressure (AP). (b) Epithelial‐mesenchymal transition (EMT) marker expression showed a significant decrease in E‐cadherin ( p = 0.031) and an increase in alpha‐smooth muscle actin (αSMA) ( p = 0.031) in the NP group; vimentin expression also tended to increase ( p = 0.051). n = 12 for each group. Values are presented as means ± standard deviations. Statistical significance is indicated as * p < 0.05 and ** p < 0.01. αSMA: Alpha‐smooth muscle actin, AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HIF‐1α: hypoxia‐inducible factor‐1 alpha, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, TGF‐β1: transforming growth factor‐beta 1, THBS1: thrombospondin‐1.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Marker, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Gene expression in PSVK1 keratinocytes exposed to negative‐pressure and THBS1 inhibition. Real‐time polymerase chain reaction (PCR) was performed on cultured human keratinocyte PSVK1 cells exposed to ambient pressure (AP) or negative‐pressure (NP), with or without treatment with Leucine‐Serine‐Lysine‐Leucine (LSKL), a thrombospondin‐1 (THBS1) inhibitor or dimethyl sulfoxide (DMSO), a vehicle control. TaqMan assays revealed that THBS1 expression was reduced under NP when treated with LSKL. Transforming growth factor‐beta 1 ( TGFB1 ) expression also decreased with LSKL under AP. Cadherin 1 ( CDH1 ), which encodes E‐cadherin, was downregulated under NP regardless of treatment. Vimentin ( VIM ) expression increased under NP with vehicle but showed a decreasing trend with LSKL. Actin alpha 2 ( ACTA2 ), encoding αSMA, was consistently downregulated by LSKL treatment under both pressure conditions. Data represent eight replicates per group and were analysed using two‐way analysis of variance with Tukey's range test. Significance is indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001. ACTA2: actin alpha 2, αSMA: alpha‐smooth muscle actin, CDH1: Cadherin 1, DMSO: dimethyl sulfoxide, LSKL: Leucine‐Serine‐Lysine‐Leucine, PCR: polymerase chain reaction, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1, VIM: vimentin.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Gene Expression, Inhibition, Real-time Polymerase Chain Reaction, Cell Culture, Control, Expressing, Polymerase Chain Reaction

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Histological and immunohistochemical changes in NP‐treated diabetic mouse skin. (a) Immunohistochemistry (IHC) staining was performed to evaluate the epithelial‐mesenchymal transition (EMT)‐related marker thrombospondin‐1 (THBS1). THBS1‐positive pixels in the epidermis were quantified. THBS1 positivity was significantly higher in the negative‐pressure (NP) group compared with the other two groups ( p < 0.001). (b) Skin thickness was assessed in haematoxylin and eosin (HE)‐stained sections. Both epidermal and dermal thickness were significantly greater in the NP group than in the other two groups ( p < 0.001). (c) IHC staining for hypoxia‐inducible factor‐1 alpha (HIF‐1α), plasminogen activator inhibitor‐1 (PAI‐1), and fibrinogen was performed to evaluate tissue hypoxia and microthrombosis induced by NP. (c1) PAI‐1 positivity in the epidermis was significantly higher in the NP group ( p < 0.001). (c2) The number of HIF‐1α positive cells per mm 2 in the epidermis was significantly increased in the NP group ( p < 0.001). (c3) The number of fibrinogen‐positive vessels per mm 2 in the dermis was significantly higher in the NP group ( p < 0.001). Arrowheads indicate fibrinogen‐positive vessels. (d) Angiogenesis was evaluated using IHC for CD31. The number of CD31‐positive vessels per mm 2 in the dermis was significantly higher in the NP group ( p < 0.001). Arrowheads indicate CD31‐positive vessels. N = 6 each. Values are presented as means ± standard deviations. Statistical significance is indicated as *** p < 0.001. Bar: 50 μm. AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HE: haematoxylin and eosin, HIF‐1α: hypoxia‐inducible factor‐1 alpha, IHC: immunohistochemistry, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Immunohistochemical staining, Immunohistochemistry, Marker, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Quantitative analysis of EMT‐ and angiogenesis‐related proteins in NP‐treated mouse skin. Enzyme‐linked immunosorbent assay (ELISA) of skin tissue samples revealed significantly increased levels of thrombospondin‐1 (THBS1), transforming growth factor‐beta 1 (TGF‐β1), plasminogen activator inhibitor‐1 (PAI‐1), hypoxia‐inducible factor‐1 alpha (HIF‐1α) and vascular endothelial growth factor (VEGF) in the negative‐pressure (NP) group compared with the control and ambient pressure (AP) groups. THBS1: p = 0.012 versus control, p = 0.011 versus AP; TGF‐β1: p = 0.008 versus control, p = 0.006 versus AP; PAI‐1: p < 0.001 versus both; HIF‐1α: p = 0.004 versus control, p = 0.001 versus AP; VEGF: p < 0.001 versus both. N = 6 per group. Values are presented as means ± standard deviations. Statistical significance is indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001. AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HIF‐1α: hypoxia‐inducible factor‐1 alpha, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1, VEGF: vascular endothelial growth factor.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Enzyme-linked Immunosorbent Assay, 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: 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: 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: 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: 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