recombinant mouse thbs4 protein  (R&D Systems)

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