pfak 7397 d20b1 cell signaling cat  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Non-cancer stem cell-derived Fibromodulin activates Integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth

doi: 10.1101/2022.04.03.486893

Figure Lengend Snippet: A. Heatmap showing differentially regulated proteins in endothelial cells (treated with vehicle or 400 nM rhFMOD for 10, 30 and 60 min, Log 2 fold change >/< 0.2), assessed by RPPA (Reverse Phase Protein Array). Red and green depict upregulated and downregulated proteins in ST1 cells, respectively. The red arrows indicate HES1 and pFAK. B. qRT-PCR analysis shows that rhFMOD treatment of ST1 cells causes a time-dependent increase in HES1 mRNA, which is inhibited in cells pre-treated with GSI (10 μM). C. Western blotting showing rhFMOD treatment of ST1 cells causes an increase in HES1 protein, which is inhibited in cells pre-treated with GSI. D. Quantification of the number of networks formed in in vitro angiogenesis assay shows that the rhFMOD-mediated increase in the number of networks formed by ST1 cells is abolished in cells pre-treated with GSI. E. Western blotting shows a time-dependent increase in phospho-FAK level in FMOD-treated ST1 cells. This increase is suppressed when the cells are pre-treated with RGD peptide (10 μM), an Integrin inhibitor. F. Quantification of networks upon treatment of ST1 cells with either LN229/Vector CM or LN229/FMOD CM. ST1 cells are pretreated with the indicated inhibitors (PP2, PP3, and FAK inhibitor-PF573228 were used at a concentration of 10 μM, ROCK1 inhibitor-H1152 was used at a concentration of 0.5 μM, Rac1 inhibitor was used at a concentration of 10 μM). G. qRT-PCR analysis shows that rhFMOD treatment of ST1 cells causes a time-dependent increase in HES1 mRNA, which is inhibited in cells are pre-treated with RGD peptide. H. Western blotting showing rhFMOD treatment of ST1 cells causes an increase in HES1 protein level, which is inhibited in cells pre-treated with the RGD peptide. I . Western blotting validating the knockdown of JAG1 in shJAG1-transfected ST1 cells. J. qRT-PCR analysis shows that rhFMOD-induced expression of HES1 mRNA is significantly decreased in ST1/shJAG1 cells compared with ST1/shNT cells. K. rhFMOD-induced expression of HES1 protein is decreased in ST1/shJAG1 cells compared with ST1/shNT cells. L. Quantification of networks formed in in vitro angiogenesis assay upon treatment of ST1/shNT and ST1/shJAG1 cells with BSA or rhFMOD. For panels B, D, F, G, J, and L, the p-value calculated by unpaired t-test with Welch’s correction are indicated. p-value less than 0.05 was considered significant with *, **, *** representing p-value less than 0.05, 0.01 and 0.001 respectively.

Article Snippet: FMOD (Abgent AP9243b, 1:2000), FAK (Cell Signaling Technology 3285S, 1:1000), pFAK (Cell Signaling Technology #3283S, 1:500), GAPDH (Sigma #G8795, 1:20,000), Actin (Sigma A3854, 1:20,000), HES1 (Cell Signaling Technology #D6P2U, 11988S, 1:1000), vWF (Abcam #6994, 1:2000), CD31 (Cell Signaling Technology #89C2, Mouse mAb 1:200 for IHC), JAG1 (Cell Signaling Technology, Jagged1 (D4Y1R) XP ® Rabbit mAb #70109, 1:200 for IHC, 1:1000 for WB), FMOD (Fibromodulin Polyclonal Antibody PA5-26250, Invitrogen, IHC 1:100, WB 1:1000), CD133 (Recombinant Anti-CD133 antibody-EPR20980-104 #ab216323 abcam, Flow Cyt.

Techniques: Protein Array, Quantitative RT-PCR, Western Blot, In Vitro, Angiogenesis Assay, Plasmid Preparation, Concentration Assay, Knockdown, Transfection, Expressing

Journal: bioRxiv

Article Title: Non-cancer stem cell-derived Fibromodulin activates Integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth

doi: 10.1101/2022.04.03.486893

Figure Lengend Snippet: A. Bar diagram showing that rhFMOD cannot activate Notch-dependent reporter luciferase activity of CSL Luc in ST1 cells in the presence of WT peptide but can do so in the presence of mutant peptide. B. Bar diagram showing that rhFMOD cannot activate Notch-dependent reporter luciferase activity of HES Luc in ST1 cells in the presence of WT peptide but can do so in the presence of mutant peptide. C. Bar diagram quantifying the number of networks formed in in vitro angiogenesis assay shows that the WT (wild-type) peptide competes with rhFMOD to bind to Type I Collagen, whereas the mutant peptide cannot. Hence, rhFMOD induces more angiogenesis in the presence of the mutant peptide, as Type I Collagen is free for it to bind. However, because of the competition with the WT peptide, rhFMOD less efficiently induces angiogenesis in endothelial cells. D. Western blotting shows reduced induction in pFAk levels in rhFMOD-treated ST1/shITGB1 cells compared with the rhFMOD-treated ST1/shNT cells, where pFAK levels increae at 30 and 60 mins, while total FAK remains constant in both the cases. E. Western blotting shows reduced induction in pFAk levels in rhFMOD-treated ST1/shITGAV cells compared with the rhFMOD-treated ST1/shNT cells, where pFAK levels increae at 30 and 60 mins, while total FAK remains constant in both the cases. F. Western blotting shows reduced induction in pFAk levels in rhFMOD-treated ST1/shITGA6 cells compared with the rhFMOD-treated ST1/shNT cells, where pFAK levels increae at 30 and 60 mins, while total FAK remains constant in both the cases. G. Western blotting showing knockdown of ITGB1 in shITGB1 transduced ST1 cells compared with ST1/shNT cells. H. Western blotting showing knockdown of ITGAV in shITGAV transduced ST1 cells compared with ST1/shNT cells. I. Western blotting showing knockdown of ITGA6 in shITGA6 transduced ST1 cells compared with ST1/shNT cells. p values calculated by unpaired t test with Welch’s correction are indicated. p value less than 0.05 is considered significant with *, **, *** representing p value less than 0.05, 0.01 and 0.001 respectively. ns stands for non-significant.

Article Snippet: FMOD (Abgent AP9243b, 1:2000), FAK (Cell Signaling Technology 3285S, 1:1000), pFAK (Cell Signaling Technology #3283S, 1:500), GAPDH (Sigma #G8795, 1:20,000), Actin (Sigma A3854, 1:20,000), HES1 (Cell Signaling Technology #D6P2U, 11988S, 1:1000), vWF (Abcam #6994, 1:2000), CD31 (Cell Signaling Technology #89C2, Mouse mAb 1:200 for IHC), JAG1 (Cell Signaling Technology, Jagged1 (D4Y1R) XP ® Rabbit mAb #70109, 1:200 for IHC, 1:1000 for WB), FMOD (Fibromodulin Polyclonal Antibody PA5-26250, Invitrogen, IHC 1:100, WB 1:1000), CD133 (Recombinant Anti-CD133 antibody-EPR20980-104 #ab216323 abcam, Flow Cyt.

Techniques: Luciferase, Activity Assay, Mutagenesis, In Vitro, Angiogenesis Assay, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Non-cancer stem cell-derived Fibromodulin activates Integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth

doi: 10.1101/2022.04.03.486893

Figure Lengend Snippet: A. Immunohistochemical analysis shows co-staining of CD31 with FMOD, pFAK, JAG1, and HES 1 in brain section derived from MGG8/shNT groups of animals. B. Immunohistochemical analysis shows co-staining of CD31 with FMOD, pFAK, JAG1, and HES 1 in brain section derived from AGR53-GSC/miRNT group of animals, after doxycycline injection. C. Immunohistochemical analysis shows co-staining of CD31 with FMOD, pFAK, JAG1, and HES 1 in brain section derived from DBT-Luc-GSC/miRFMOD group of animals, without doxycycline injection. Magnification = 20x, Scale=100 μm.

Article Snippet: FMOD (Abgent AP9243b, 1:2000), FAK (Cell Signaling Technology 3285S, 1:1000), pFAK (Cell Signaling Technology #3283S, 1:500), GAPDH (Sigma #G8795, 1:20,000), Actin (Sigma A3854, 1:20,000), HES1 (Cell Signaling Technology #D6P2U, 11988S, 1:1000), vWF (Abcam #6994, 1:2000), CD31 (Cell Signaling Technology #89C2, Mouse mAb 1:200 for IHC), JAG1 (Cell Signaling Technology, Jagged1 (D4Y1R) XP ® Rabbit mAb #70109, 1:200 for IHC, 1:1000 for WB), FMOD (Fibromodulin Polyclonal Antibody PA5-26250, Invitrogen, IHC 1:100, WB 1:1000), CD133 (Recombinant Anti-CD133 antibody-EPR20980-104 #ab216323 abcam, Flow Cyt.

Techniques: Immunohistochemical staining, Staining, Derivative Assay, Injection

Journal: Frontiers in Endocrinology

Article Title: CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta

doi: 10.3389/fendo.2020.597549

Figure Lengend Snippet: Primary and secondary antibodies used for western blotting.

Article Snippet: pFAK , rabbit monoclonal , Cell Signaling (#4060S) , 1:1000 , 1:2500.

Techniques: Western Blot, Concentration Assay