Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in human pancreatic tumors. ( A ) Representative images of human pancreatic cancer (representative from n = 25 PDAC patients) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in human PDAC tumors. ( C ) Endoglin mRNA expression by human cells; ECRF endothelial cells, MIA PaCa-2, PANC-1 and BxPC-3 PDAC cells and 8 patient derived primary pancreatic CAFs. ( D ) Endoglin protein expression on human pancreatic fibroblasts. Basal and BMP9-induced downstream signaling (pSMAD1) was inhibited with TRC105 (full-length blot shown in Supplementary figure 4A – C ).

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Staining, Expressing, Double Staining, Derivative Assay

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in mouse pancreatic tumors. ( A ) Representative images of mouse pancreatic tumors (KPC) (representative from n = 5) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in mouse KPC tumors. ( C ) Endoglin mRNA expression by mouse cells; 2H11 endothelial cells, MC38 colorectal cancer, and KPC-3 pancreatic cancer cells, CAFs isolated from colorectal and pancreatic tumors.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Staining, Expressing, Double Staining, Isolation

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: TRC105 does not affect tumor growth in a murine KPC-3 model for pancreatic cancer. ( A ) Tumor volume in mm 3 and ( B ) tumor weight upon 13 days of therapy (28 days after tumor inoculation, n = 7 animals per group). ( C ) Percentage of intratumoral CD45+ cells (gated from live gate) by using flow cytometry. ( D ) Percentage of CD8+ T-cells (from CD45 gate, n = 6-7 mice per group. ( E ) Representative histological images and quantifications of endoglin ( F ), vimentin ( G ) and α-SMA ( H ) (n = 7 animals per group). ( I ) Intratumoral TRC105 levels in tumor lysates determined by ELISA (n = 5 control, n = 3 TRC105). All graphs represent mean ± SD. Student’s T -test was performed to calculate differences indicated in the graphs *p = <0.05 **p = <0.01.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Early treatment with TRC105 does not affect tumor growth but changes the tumor microenvironment. ( A ) Tumor volume in mm 3 upon 27 days of therapy and 28 days after tumor inoculation (n = 5-8 mice per group). ( B ) Percentage of infiltrating immune cells (CD45+). ( C ) CD3+, ( D ) CD8+ and ( E ) CD4+ cells out of CD45 gate. ( F ) Intratumoral CD4+ CD25+ Treg-like cells out of CD4 gate (n = 5-8 mice per group). ( G ) Heatmap summarizing qPCR data normalized to the control group of different cytokines, growth factors and stromal markers (n = 5-8 mice per group). ( H ) Representative histological pictures of α-SMA, endoglin, cytokeratin and vimentin staining (n = 5-8 mice per group). All graphs represent mean ± SD. One-way ANOVA was used to calculate statistical differences. *p = <0.05.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Control, Staining

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Col1a1 specific endoglin knock-out does not affect tumor growth but alters immune cell composition. ( A ) Tumor volume in mm 3 after 28 days of tumor inoculation (n = 7 mice per group). ( B ) Representative pictures of histological samples stained with α-SMA and endoglin (n = 6 mice per group). ( C ) CD45+ immune infiltrate and ( D ) CD3 + T-cells (from CD45+ gate). ( E ) CD8+ and ( F ) CD4+ cells from (from CD3+ gate). ( G ) Percentage CD8+ PD1+ cells (from CD8+ gate). ( H ) Percentage of CD8+ LAG-3+ cells (from CD8+ gate) (n = 6 mice per group). ( I ) Heatmap summarizing qPCR data normalized to the control group of different cytokines growth factors and stromal markers (n = 6 mice per group) ND in the graph indicates not-detectable. All graphs represent mean ± SD. Student’s T -test was performed to calculate significances indicated in the graphs **p = <0.01.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Knock-Out, Staining, Control

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Protein–protein association between galectin-3 and endoglin. ( A – C ). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. ( A ) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin ( B ) or anti-galectin-3 ( C ) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b ( B ) and IgG1 ( C ) were included. ( D ) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Article Snippet: Two replicate analyses were hybridized with purified recombinant human soluble endoglin (sEng; Glu26-Gly586; 1097-EN, R&D Systems).

Techniques: Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Control, Protein-Protein interactions, Recombinant, Protein Binding, Negative Control

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Protein–protein association between TRIM21 and endoglin. ( A – E ) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin ( A ). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin ( B ). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL ( E ) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin ( C ). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included ( D ). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. ( F ) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Article Snippet: Two replicate analyses were hybridized with purified recombinant human soluble endoglin (sEng; Glu26-Gly586; 1097-EN, R&D Systems).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Negative Control, Transfection, Expressing, Protein-Protein interactions, Recombinant, Protein Binding

Journal: STAR protocols

Article Title: Enzyme-free isolation of mesenchymal stem cells from decidua basalis of the human placenta.

doi: 10.1016/j.xpro.2023.102498

Figure Lengend Snippet: Figure 2. Immunophenotypic characterization of isolated PL-MSCs The antibodies used were (A) human CD73, (B) human CD90 (C) human CD105 (D) human CD166 (E) human CD34 (F) human HLA-DR and their respective isotype controls. Open histogram indicates background signal, while, shaded histogram represents positive reactivity with the indicated antibodies. Table shows percentage of cells expressing the respective surface markers (n = 3) in mean G SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies PE Mouse Anti-Human CD73 (1:33) BD Pharmingen Catalog No 550257 PE Mouse Anti-Human CD90 (1:33) BD Pharmingen Catalog No 555596 CD105 PE-conjugated Antibody (1:50) R&D Systems Catalog No FAB10971P PE Mouse Anti-Human CD34 (1:50) BD Pharmingen Catalog No 550761 PE Mouse Anti-Human CD166 (1:50) BD Pharmingen Catalog No 559263 FITC Mouse Anti-Human HLA-DR (1:33) BD Pharmingen Catalog No 555811 PE Mouse IgG, k Isotype Control (1:33 as isotype for CD90, 1:800 as isotype for CD34/ CD73/CD166) BD Pharmingen Catalog No 550617 Mouse IgG1 PE-conjugated Antibody (1:50) R&D Systems Catalog No IC002P FITC Mouse IgG2a, k Isotype Control (1:33) BD Pharmingen Catalog No 555573 Vimentin (D21H3) XP Rabbit mAb Antibody (1:150) Cell Signaling Technology Catalog No 5741S Human STRO-1 antibody (1:100) R&D Systems Catalog No MAB1038 Oct-4 (D705Z) Mouse mAb (1:400) Cell Signaling Technology Catalog No 75463S Alexa Fluor 488 goat anti-mouse (1:500) Invitrogen Catalog No A11001 Alexa Fluor 568 goat anti-rabbit (1:500) Invitrogen Catalog No A11011 Alexa Fluor 488 anti-rabbit (1:500) Cell Signaling Technology Catalog No 4412S Biological samples Human placenta N/A N/A Chemicals, peptides, and recombinant proteins 0.9% w/v Saline Kunal Remedies Pvt.

Techniques: Isolation, Expressing