anti thbs4 sheep anti mouse  (R&D Systems)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 expression in human skin following burn injury. (A) Normal skin from healthy controls; (B) skin from burn injury patients. Biopsy samples were collected from the study area at 3, 14, and 21 days after the wound excision. E—epidermis. 3 representative samples in each group are shown. Scale bar is 200 μm. (C) Relative quantification of THBS4 expression by mean integrated density of the fluorescence signal. The plot depicts the distribution of 10 samples, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Expressing, Quantitative Proteomics, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 is upregulated in regenerating mouse skin. Full-thickness dermal wounds were generated in mouse dorsal skin. (A) THBS4 expression was characterized by immunofluorescence microscopy in healthy skin and at 2-, 4-, 6-, and 8-days post wounding. 3 representative samples in each group are shown. Yellow arrows indicate THBS4 expression in hair follicles. Scale bar is 200 μm. (B) Relative quantification of THBS4 expression by mean integrated density of the fluorescence signal. Bars show the average of 3 samples for each time point ± standard deviation, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Generated, Expressing, Immunofluorescence, Microscopy, Quantitative Proteomics, Fluorescence, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 expression in healthy control skin and psoriatic skin lesions. E—epidermis; D—dermis. 3 representative samples in each group are shown (A) and relative quantification of THBS4 expression by mean integrated density of the fluorescence signal (B) , n = 5. Scale bar is 200 μm. (C) THBS4 co-localization with fibroblast marker vimentin (Vim) and integrin beta 4 (ITGB4). Scale bar is 50 μm. The plot depicts the distribution of 5 samples, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Expressing, Control, Quantitative Proteomics, Fluorescence, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes fibroblast migration. (A) Transwell migration assay with fibroblasts stimulated with recombinant THBS4 and quantification of the number of migrating cells in a field of view. (B) Transwell invasion assay through Matrigel-coated chambers and quantification of the number of migrating cells in a field of view. (C) Representative images of the in vitro scratch wound healing assay with fibroblasts stimulated with recombinant THBS4 and quantification of the relative wound closure in time. Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference compared to cells stimulated with control medium.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Migration, Transwell Migration Assay, Recombinant, Transwell Invasion Assay, In Vitro, Wound Healing Assay, Standard Deviation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes keratinocyte but not fibroblast proliferation in vitro . (A) Representative images of primary human fibroblasts cultured in the presence of recombinant THBS4 protein and the quantification of Ki67 + positive cells, n = 3 (B) . (C) Representative images of primary human keratinocytes cultured in the presence of recombinant THBS4 protein and quantification of Ki67 + positive cells. (D) Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference compared to cells stimulated with control medium.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: In Vitro, Cell Culture, Recombinant, Standard Deviation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Heatmap of differentially enriched genes in fibroblasts in response to THBS4 stimulation. Statistically significant ( P < 0.05) and expression fold-change > 2 protein coding genes and microRNAs are shown, n = 2.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Ingenuity Pathway Analysis of the changes in fibroblast transcription profile in response to THBS4 stimulation. Causal network showing Frizzled and β-catenin 1 pathway activation is shown. Upper values indicate log2 fold changes, lower values show the P -value for the RNAseq.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Proteotranscriptomic analysis of THBS4 stimulation in fibroblasts. The combined network of transcriptomics analysis at 4 h stimulation and proteomics analysis at 24 h is shown. Single numbers indicate log2FC in proteomics and duplicate values indicate log2FC and P -value for RNAseq.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes wound healing in vivo . Full dermal wounds were inflicted on the dorsal skin of C57/Bl6 mice and either recombinant THBS4 or PBS was applied. Representative images of the wounds from indicated time points (A) and measurements of the wound area (B) . The graph depicts the averages of 10 biological replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: In Vivo, Recombinant, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 is a soluble dermal inflammatory signal that activates the fibroblast migration for skin regeneration and wound healing. See section “Discussion” for closer explanation.

Article Snippet: In experiments testing the effect of THBS4 protein on the wound healing process, 1 μg of purified recombinant mouse THBS4 protein (R&D Systems, Minneapolis, MN, United States), product code 7860-TH-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily on the wounds.

Techniques: Migration

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: BM-MSC engraftment leads to THBS4 overexpression during H. pylori infection. ( A ) Volcano plot presentation of differentially expressed genes of gastric tissues in H. pylori versus H. pylori + MSCs mice. ( B ) Enrichment analysis of differential genes. ( C ) RT-PCR verified the expression of differential genes associated with angiogenesis. THBS4 was the most highly expressed gene. n= 10 mice in each group. ( D , E ) Western blot analysis showed that BM-MSC transplantation led to THBS4 overexpression. n= 3 mice in each group. ( F , G ) IHC ( F ) and IF ( G ) of THBS4 in H. pylori + MSCs or H. pylori mice. n= 3 mice per group Cell nuclei, DAPI. THBS4, FITC. Magnification: × 200 and × 400. ( H ) Quantification of F. ( I , J ) Western blot analysis of THBS4 revealed that H. pylori pretreatment increased the protein expression of THBS4 in BM-MSCs. n= 3 wells per group.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: Over Expression, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transplantation Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Overexpression of THBS4 is correlated with vessel density in human GC tissues and predicts a poor prognosis. ( A ) The expression of THBS4 in a pan-cancer dataset. The red and green labels at the top represent high and low expression in certain tumor types, respectively. ( B ) Volcano plot revealed upregulation of THBS4 mRNA expression in GC compared with normal tissue. ( C , D ) The overall survival and disease-free survival analyses of THBS4 were plotted for patients with GC in TCGA. ( E ) Representative IHC staining with THBS4 antibody in GC and adjacent nontumor tissues. n= 5 samples per group. ( F ) Quantification of E. ( G ) Representative IHC staining with PECAM1 antibody in GC and adjacent nontumor tissues. n= 5 samples per group. ( H ) Quantification of G. ( I ) Correlation analysis of THBS4 and vessel density showed that THBS4 was positively correlated with vessel density in GC. ( J ) Validation of the positive correlation between THBS4 and PECAM1 in GEPIA.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: Over Expression, Expressing, Immunohistochemistry, Biomarker Discovery

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: THBS4 mediates BM-MSCs to promote the migration and tube formation of HUVECs in vitro . ( A ) Transfection of recombinant lentiviral vectors into BM-MSCs. ( B – E ) qRT-PCR and Western blot analysis of THBS4 expression in BM-MSCs of both experiments transfected as indicated. n= 3 wells per group. ( F , H ) Transwell assays and quantification of migrated cells showed that THBS4 knockdown in BM-MSCs inhibited their ability to promote HUVEC migration. n= 3 wells in each group. ( G , I ) Knockdown of THBS4 in BM-MSCs alleviated its ability to promote HUVEC tube formation. n= 5 wells per group.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: Migration, In Vitro, Transfection, Recombinant, Quantitative RT-PCR, Western Blot, Expressing, Knockdown

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: THBS4 mediates BM-MSC-induced promotion of GC angiogenesis in vivo . ( A , B ) Four-week-old nude male mice were injected with either SGC cells only or SGC and BM-MSCs transfected with recombinant lentiviral vectors. Three weeks following injection, the mice were euthanized, and the tumors were analyzed. n= 3 mice in control group and 5 mice per group in other groups. ( C ) Growth curve of tumors described in A. ( D ) Tumor weight of injected tumors at end-point. ( E ) H&E staining and Ki-67 and CD31 immunostaining of tumors described in A. ( F , G ) Quantification of staining presented in E performed with Image Plus. ( H , I ) Following AngioSense 750EX injection, tumor vascular permeability fluorescence imaging in vivo was performed to detect the neovascularization density of tumors in A. ( J , K ) Representative images and quantification of the CAM assay. n= 5 eggs in each group. ( L, M ) Western blot analysis of THBS4 in gastric tissues after NC BM-MSC or THBS4-knockdown BM-MSC transplantation. n= 3 mice in each group. ( N ) IF of THBS4 in gastric tissues after NC BM-MSC or THBS4-knockdown BM-MSC transplantation. ( O ) IF of CD31 and NG2 in gastric tissues in NC BM-MSC-transplanted mice and THBS4-knockdown BM-MSC-transplanted mice. n= 10 mice in each group.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: In Vivo, Injection, Transfection, Recombinant, Control, Staining, Immunostaining, Permeability, Fluorescence, Imaging, Chick Chorioallantoic Membrane Assay, Western Blot, Knockdown, Transplantation Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Integrin α2 mediates the effect of paracrine THBS4 signaling on the migration and tube formation of HUVECs. ( A – C ) Western blotting and IF showed that integrin α2 in HUVECs was significantly increased by rTHBS4. n= 3 wells per group. ( D ) Representative images of the migration assay after rTHBS4, rTHBS4 + anti- α2 or anti- α2 treatments in HUVECs. n= three independent experiments. ( E ) Representative images of the tube formation assay after rTHBS4, rTHBS4 + anti-α2 or anti-α2 treatments in HUVECs. n= 3 wells in each group. ( F ) Quantification of D. ( G ) Quantification of E. ( H ) CCK-8 assay to detect HUVEC proliferation after rTHBS4, rTHBS4 + anti-α2 or anti-α2 treatments. n= three independent experiments.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: Migration, Western Blot, Tube Formation Assay, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Activation of AKT by THBS4/integrin α2 axis in HUVECs. ( A , B ) rTHBS4 induced the phosphorylation of AKT in HUVECs. n= three independent experiments. The HUVECs were treated with and without rTHBS4 for 1h and the whole cell lysates were analyzed by WB for levels of downstream kinases. ( C , D ) p-AKT activation occurred in a time-dependent manner in rTHBS4-treated cells. n= three independent experiments. ( E , F ) The HUVECs were treated with rTHBS4 for 1h in the presence of integrin α2-specific antibody or PI3K inhibitor LY294002. Western blot analysis showed that rTHBS4 increased Akt phosphorylation in HUVECs, while this effect was alleviated by blocking integrin α2 or inhibiting Akt. n= three independent experiments. ( G ) Transwell migration assay to investigate the effect of PI3K inhibition and integrin α2 blockade on HUVEC migration after treatment with rTHBS4. n= 3 wells per group. ( H ) In vitro tube formation to investigate the effect of PI3K inhibition and blocking integrin α2 on HUVEC tube formation. n= 3 wells per group. ( I ) Quantification of G. ( J ) Quantification of H. ( K , L ) The CM from BM-MSCs stimulated by H. Pylori was applied to HUVECs in the presence or absence of integrin α2-specific antibody or PI3K inhibitor LY294002 for Transwell migration assay and tube formation assay. n= 3 wells per group. ( M ) Quantification of K. ( N ) Quantification of L.

Article Snippet: The primary antibodies used were sheep anti-mouse THBS4 (AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-phospho-AKT (p-AKT) (Ser473) (#4060T, CST).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Transwell Migration Assay, Inhibition, Migration, In Vitro, Tube Formation Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: BM-MSC engraftment leads to THBS4 overexpression during H. pylori infection. ( A ) Volcano plot presentation of differentially expressed genes of gastric tissues in H. pylori versus H. pylori + MSCs mice. ( B ) Enrichment analysis of differential genes. ( C ) RT-PCR verified the expression of differential genes associated with angiogenesis. THBS4 was the most highly expressed gene. n= 10 mice in each group. ( D , E ) Western blot analysis showed that BM-MSC transplantation led to THBS4 overexpression. n= 3 mice in each group. ( F , G ) IHC ( F ) and IF ( G ) of THBS4 in H. pylori + MSCs or H. pylori mice. n= 3 mice per group Cell nuclei, DAPI. THBS4, FITC. Magnification: × 200 and × 400. ( H ) Quantification of F. ( I , J ) Western blot analysis of THBS4 revealed that H. pylori pretreatment increased the protein expression of THBS4 in BM-MSCs. n= 3 wells per group.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: Over Expression, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transplantation Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Overexpression of THBS4 is correlated with vessel density in human GC tissues and predicts a poor prognosis. ( A ) The expression of THBS4 in a pan-cancer dataset. The red and green labels at the top represent high and low expression in certain tumor types, respectively. ( B ) Volcano plot revealed upregulation of THBS4 mRNA expression in GC compared with normal tissue. ( C , D ) The overall survival and disease-free survival analyses of THBS4 were plotted for patients with GC in TCGA. ( E ) Representative IHC staining with THBS4 antibody in GC and adjacent nontumor tissues. n= 5 samples per group. ( F ) Quantification of E. ( G ) Representative IHC staining with PECAM1 antibody in GC and adjacent nontumor tissues. n= 5 samples per group. ( H ) Quantification of G. ( I ) Correlation analysis of THBS4 and vessel density showed that THBS4 was positively correlated with vessel density in GC. ( J ) Validation of the positive correlation between THBS4 and PECAM1 in GEPIA.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: Over Expression, Expressing, Immunohistochemistry, Biomarker Discovery

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: THBS4 mediates BM-MSCs to promote the migration and tube formation of HUVECs in vitro . ( A ) Transfection of recombinant lentiviral vectors into BM-MSCs. ( B – E ) qRT-PCR and Western blot analysis of THBS4 expression in BM-MSCs of both experiments transfected as indicated. n= 3 wells per group. ( F , H ) Transwell assays and quantification of migrated cells showed that THBS4 knockdown in BM-MSCs inhibited their ability to promote HUVEC migration. n= 3 wells in each group. ( G , I ) Knockdown of THBS4 in BM-MSCs alleviated its ability to promote HUVEC tube formation. n= 5 wells per group.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: Migration, In Vitro, Transfection, Recombinant, Quantitative RT-PCR, Western Blot, Expressing, Knockdown

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: THBS4 mediates BM-MSC-induced promotion of GC angiogenesis in vivo . ( A , B ) Four-week-old nude male mice were injected with either SGC cells only or SGC and BM-MSCs transfected with recombinant lentiviral vectors. Three weeks following injection, the mice were euthanized, and the tumors were analyzed. n= 3 mice in control group and 5 mice per group in other groups. ( C ) Growth curve of tumors described in A. ( D ) Tumor weight of injected tumors at end-point. ( E ) H&E staining and Ki-67 and CD31 immunostaining of tumors described in A. ( F , G ) Quantification of staining presented in E performed with Image Plus. ( H , I ) Following AngioSense 750EX injection, tumor vascular permeability fluorescence imaging in vivo was performed to detect the neovascularization density of tumors in A. ( J , K ) Representative images and quantification of the CAM assay. n= 5 eggs in each group. ( L, M ) Western blot analysis of THBS4 in gastric tissues after NC BM-MSC or THBS4-knockdown BM-MSC transplantation. n= 3 mice in each group. ( N ) IF of THBS4 in gastric tissues after NC BM-MSC or THBS4-knockdown BM-MSC transplantation. ( O ) IF of CD31 and NG2 in gastric tissues in NC BM-MSC-transplanted mice and THBS4-knockdown BM-MSC-transplanted mice. n= 10 mice in each group.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: In Vivo, Injection, Transfection, Recombinant, Control, Staining, Immunostaining, Permeability, Fluorescence, Imaging, Chick Chorioallantoic Membrane Assay, Western Blot, Knockdown, Transplantation Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Integrin α2 mediates the effect of paracrine THBS4 signaling on the migration and tube formation of HUVECs. ( A – C ) Western blotting and IF showed that integrin α2 in HUVECs was significantly increased by rTHBS4. n= 3 wells per group. ( D ) Representative images of the migration assay after rTHBS4, rTHBS4 + anti- α2 or anti- α2 treatments in HUVECs. n= three independent experiments. ( E ) Representative images of the tube formation assay after rTHBS4, rTHBS4 + anti-α2 or anti-α2 treatments in HUVECs. n= 3 wells in each group. ( F ) Quantification of D. ( G ) Quantification of E. ( H ) CCK-8 assay to detect HUVEC proliferation after rTHBS4, rTHBS4 + anti-α2 or anti-α2 treatments. n= three independent experiments.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: Migration, Western Blot, Tube Formation Assay, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: THBS4/integrin α2 axis mediates BM-MSCs to promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infection

doi: 10.18632/aging.203334

Figure Lengend Snippet: Activation of AKT by THBS4/integrin α2 axis in HUVECs. ( A , B ) rTHBS4 induced the phosphorylation of AKT in HUVECs. n= three independent experiments. The HUVECs were treated with and without rTHBS4 for 1h and the whole cell lysates were analyzed by WB for levels of downstream kinases. ( C , D ) p-AKT activation occurred in a time-dependent manner in rTHBS4-treated cells. n= three independent experiments. ( E , F ) The HUVECs were treated with rTHBS4 for 1h in the presence of integrin α2-specific antibody or PI3K inhibitor LY294002. Western blot analysis showed that rTHBS4 increased Akt phosphorylation in HUVECs, while this effect was alleviated by blocking integrin α2 or inhibiting Akt. n= three independent experiments. ( G ) Transwell migration assay to investigate the effect of PI3K inhibition and integrin α2 blockade on HUVEC migration after treatment with rTHBS4. n= 3 wells per group. ( H ) In vitro tube formation to investigate the effect of PI3K inhibition and blocking integrin α2 on HUVEC tube formation. n= 3 wells per group. ( I ) Quantification of G. ( J ) Quantification of H. ( K , L ) The CM from BM-MSCs stimulated by H. Pylori was applied to HUVECs in the presence or absence of integrin α2-specific antibody or PI3K inhibitor LY294002 for Transwell migration assay and tube formation assay. n= 3 wells per group. ( M ) Quantification of K. ( N ) Quantification of L.

Article Snippet: After dewaxing and hydration, the sections were incubated overnight with antibodies against human THBS4 (YT4646, ImmunoWay), human PECAM-1 (ab9498, Abcam), mouse CD31 (AF3628-SP, R&D Systems), mouse NG2 (sc-5389, Santa Cruz), mouse THBS4 (AF7860-SP R&D Systems), and mouse Ki-67 (ab1667, Abcam) and identified with Alexa 488, Alexa 594 or Cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Transwell Migration Assay, Inhibition, Migration, In Vitro, Tube Formation Assay

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 reduces surface integrin levels and destabilizes the sarcolemma. a Representative Western blots of sarcolemma protein extracts from hearts of tTA control, Thbs3 DTG, and Thbs4 DTG mice, assayed for integrin proteins and β-dystroglycan. Laminin2α2 and Cacna1c serve as loading controls. Quantitation is shown in Supplementary Figure . The red box shows integrin proteins specifically reduced on the sarcolemma by Thbs3. b Western blots for input of microsomal protein extracts before immunoprecipitation for Thbs3 and β1D integrin and Cacna1c from NRVMs infected with Adβgal or AdThbs3 (left panel). The microsomal fraction was then immunoprecipitated (IP) for β1 integrin and blotted for the indicated proteins (right panel). Mouse IgG was used as a control for the IP. c Regimen of Evans blue dye (EBD) and isoproterenol (Iso, 300 mg/kg) injection to measure membrane permeability in the hearts of mice. d Representative histological heart sections from 3 tTA control and 3 Thbs3 DTG mice, imaged for WGA (green fluorescence) and leakage of EBD into the cardiomyocytes by red fluorescence subjected to the experimental regimen shown in c . Scale bars are 1 mm. e Regimen of Evans blue dye (EBD) injection after TAC surgery to measure membrane permeability in adult mice. Mice were sacrificed (Sac) 24 h after EBD injection, 1 week after TAC surgery. f , g Representative cardiac histological images of tTA control and Thbs3 DTG hearts 1 week after TAC surgery stained with WGA (green) and intracellular leakage of EBD (red) according to the experimental scheme shown in e , while g shows quantitation of EBD positive cells in the hearts of these mice. Scale bars are 100 μm. * P < 0.05 versus tTA control sham; two-tailed students T -test. Data are represented as percentage EBD positive cells (≥3000 cells from ≥8 animals). Error bars are +/− standard error of the mean and number of mice used is shown in the graph as individual data points

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Western Blot, Control, Quantitation Assay, Immunoprecipitation, Infection, Injection, Membrane, Permeability, Fluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Integrin overexpression reduces Thbs3-mediated membrane instability and disease. a Schematic of the breeding used to generate combinatorial Thbs3 DTG, α7β1D integrin TG mice. b Representative Western blots for Thbs3 and the indicated integrin proteins from heart sarcolemma protein extracts from tTA, Thbs3 DTG, α7β1D integrin TG and Thbs3 DTG/ α7β1D integrin TG mice. The α1Na + /K + -ATPase served as loading control. c Quantification of EBD positive area from cardiac histological sections after Iso (300 mg/kg) injection in Thbs3 DTG, α7β1D integrin TG, Thbs4 DTG, and Thbs3 DTG/ α7β1D integrin TG mice. d Representative histological images of heart sections from tTA, Thbs3 DTG and Thbs3 DTG/ α7β1D integrin TG mice stained with WGA-FITC (green) after Iso injection (300 mg/kg). EBD is shown as red fluorescence. Scale bars are 300 μm. e Fractional shortening (FS) percentage as determined by echocardiography following 2 weeks of continuous Iso infusion (60 mg/kg/day) or PBS vehicle controls. Number of mice analyzed is shown within each histogram in c , e . * P < 0.05 versus vehicle treated; #<0.05 versus Thbs3 DTG with Iso. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/− standard error of the mean

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Over Expression, Membrane, Western Blot, Control, Injection, Staining, Fluorescence

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 enhances secretory pathway activity but reduces membrane integrins. a Time course of intracellular trafficking fluorescence changes in cultured neonatal ventricular cardiomyocytes (NRVMs) infected with a GalNac-T2-RFP baculovirus and the indicated adenoviruses. The data show a quantitative time course of GalNac-T2-RFP recovery in the Golgi network after FRAP to measure ER-to-Golgi vesicular trafficking. * P < 0.05 versus Adβgal infected cells. b Quantitative time course of loss of VSVG-eGFP fluorescence in the Golgi after iFRAP as a measurement for Golgi-to-membrane (Golgi exit) vesicular trafficking. * P < 0.05 versus Adβgal infected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. c Western blots for ECM proteins using heart extracellular matrix extracts from tTA, Thbs3 DTG and Thbs4 DTG mice at baseline or with TAC stimulation. A silver-stained gel is shown with a non-specific (n.s.) band as a loading control. d , e Quantitative time-course of the loss of α5 integrin-GFP fluorescence at the Golgi after iFRAP as a measure of Golgi-to-plasma membrane vesicular trafficking of α5 integrin. The data were generated by iFRAP of α5 integrin-GFP from COS-7 cells d transfected with the plasmids harboring the cDNAs shown or e treated with recombinant Thbs3 or Thbs4 proteins, or bovine serum albumin (BSA) as a control. * P < 0.05 versus Adβgal transfected cells; #<0.05 versus Thbs3 transfected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. Results were summed from four independent experiments and error bars are +/− standard error of the mean

Article Snippet: The denaturation reaction was carried out at room temperature for 40 min. Denaturated luciferase was diluted 125 fold in refolding buffer (25 mM Hepes, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM ATP) supplemented with 2 μM BSA or 2 μM of the indicated chaperone protein and incubated at room temperature for 60 min (recombinant heat shock protein 70 (Enzo Life Sciences, #ADI-NSP-555-D), recombinant Thbs4 (R&D Systems #7860-TH-050), recombinant Nell2 (Aviva Systems Biology #OPPB00457), bovine serum albumin (BSA, ThermoFisher Scientific, #BP1600), recombinant Thbs3 (R&D Systems, #8390-TH-050)).

Techniques: Activity Assay, Membrane, Fluorescence, Cell Culture, Infection, Western Blot, Staining, Control, Clinical Proteomics, Generated, Transfection, Recombinant