Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: The effect of statins on viability and growth of ( a ) stem ADMSC and non-cancerous HEK 293 cells and ( b ) cancer MiaPaCa-2 cells. ( a ) ADMSC—human adipose-derived mesenchymal stem cells, HEK 293—human embryonic kidney cells, exposure to statins—24 h, concentrations 0—100 µM, control—methanol, ( b ) previously published data , MiaPaCa-2—pancreatic cancer cells, exposure to statins—24 h, concentrations 0—40 µM, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of the effect of statins on the growth and viability of pancreatic cancer MiaPaCa-2 cells, non-cancerous HEK 293 cells, and ADMSC stem cells. Concentration of statins—20 µM, Time—exposure to statins—24, 48, and 72 h, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on size and compactness of spheroids. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr—methanol treated spheroids, P—pravastatin, R—rosuvastatin, L—lovastatin, F—fluvastatin, A—atorvastatin, Pi—pitavastatin, C—cerivastatin, S—simvastatin. Statins were added once, after spheroid formation, 10 weeks ( a ) or 3.5 weeks ( b ) after inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on the spheroid formation. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr methanol treated spheroids, P —pravastatin, R —rosuvastatin, L —lovastatin, F —fluvastatin, A —atorvastatin, Pi —pitavastatin, C —cerivastatin, S —simvastatin. Statins were added once, 24 h after cell inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of expression changes between statin treated and control MiaPaCa-2 and ADMSC cells. Displayed are only the genes that are differentially expressed upon at least one statin treatment in at least one cell type, requiring |log 2 FC|> 1 and FDR < 0.05. Statins were administered at a concentration of 12 µM for 24 h. ( FC fold change, FDR false discovery rate, horizontal and vertical axes—changes in ADMSC and MiaPaCa-2 cells, respectively, upon respective treatment). The red dashed lines indicate two-fold change increase or decrease in the gene expression. The genes with at least two-fold up-regulation (resp. down-regulation) in ADMSC stem cells are displayed to the right (resp. left) of the dashed lines. Similarly, genes with at least two-fold up-regulation (resp. down-regulation) in cancer cells are displayed above (resp. below) of the dashed lines. For details about differentially regulated transcripts see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579 .

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Expressing, Control, Concentration Assay, Gene Expression

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Cellular pathways most significantly affected by statins in cancer and stem cells. The gene set enrichment analysis (GSEA) revealed the KEGG pathways most affected by statin treatment in ADMSC and MiaPaCa-2 cells. Displayed is the union of the top five most enriched pathways among the comparisons. (Statin concentration—12 µM, treatment time—24 h, p-value—GSEA p-value, gene ratio—fraction of KEGG pathway genes among differentially expressed genes). For details about differentially regulated transcripts, see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

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

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: Cancer cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet: Figure 4. Phenotypic plasticity of mesenchymal marker expression (A) Flow cytometry analysis of PDPN and CD105 in purified and in-vitro-cultured CD105pos and CD105neg pancreatic fibroblasts (PaFs) after 1 and 7 weeks. Plots are representative of n = 4 experiments. Relative frequencies shown in relevant quadrants. (B) Normalized Eng mRNA expression in purified CD105pos (n = 4) and CD105neg PaFs (n = 4) treated with control (top) or KPC PDA conditioned medium (bottom). Data displayed as mean ± SD. (C) Representative flow cytometry analysis (n = 4) of CD105 on GFPposCD105pos and GFPposCD105neg PaFs in mono- or co-culture with RFPpos KPC PDA tu- mor cells. (D) Representative flow cytometry analysis (n = 3) of CD105 in isolated CD105pos and CD105neg human PaFs after >3 weeks of in vitro culture. (E and F) MC analysis of primary PaFs treated with the indicated ligands for 3 days. Representative plots displaying relative frequencies of CD105pos and CD105neg PaFs. (G and H) Heatmap of median marker intensity (MMI) displayed as column Z scores for each phenotypic marker on CD105pos (G) and CD105neg (H) PaFs after 3 days of treatment as indicated. Boxplots show MMI with upper and lower boundary of the interquartile range and whiskers denoting maximum and minimum values minus outliers, across all conditions. (I and J) Representative flow cytometry analysis (n = 3) of CD105pos (I) and CD105neg (J) PaFs with IFN-g, IFN-g + KPC PDA conditioned medium, or IFN-g + TGF- b1 treatment. Samples are compared using unpaired t tests (B) (top and bottom). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5 and Table S4.

Article Snippet: The first staining round used mouse anti-human CD105 antibody (CST clone 3A9) at 1/200 and TSA570 (FP1488001KT).

Techniques: Marker, Expressing, Flow Cytometry, In Vitro, Cell Culture, Control, Cytometry, Co-Culture Assay, Isolation

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: DBM-MSN/152RM scaffolds coordinate the recruitment of MSCs and ECs in vivo . (A) HE staining images demonstrating the recruitment of MSCs and ECs 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (B) Co-immunofluorescence staining of CXCR4, integrin αvβ3 and CD271 in MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (C) Immunofluorescence staining of CD90 + CD105 + MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (D) Co-immunofluorescence staining of emcn and integrin αvβ3 in type H vessels from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (E) Immunofluorescence staining of CD31 + in EPCs from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. Data are shown as the mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA was employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: In Vivo, Staining, Immunofluorescence

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: Clinical Cancer Research

Article Title: Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging

doi: 10.1158/1078-0432.ccr-05-0876

Figure Lengend Snippet: Fig. 5. In situ characterization of EGFP+ cells in HT-29 (s.c.) + TG-hMSC (i.v.) tumors. A, agarose gel electrophoresis of PCR-amplified EGFP sequence (717 bp) done using1 Ag of cellular DNA prepared from loosely associated cell fraction (loose), stromal matrix associated fraction (stroma), and the residue (residue) of HT-29 (s.c.)/TG-hMSC (i.v.) tumor and HT-29 (s.c.) tumor,TG-hMSC, and HT-29 cells. B, cytofluorometric histograms comparing the stromal matrix cell fractions of HT-29 (s.c.) + TG-hMSC (i.v.) tumors (red) and HT-29 (s.c.) tumors (black), examined with anti-GFP specific antibody. EGFP+ cells in the gated area represented 11.5% of total cells. C, histograms showing the presence of human endothelial cell markers but absence of hMSC markers in EGFP+ cells obtained from the HT-29 (s.c.) + TG-hMSC (i.v.) tumor after 30 days of growth in vivo. Stromal matrix ^ associated cell fractions were double stained with FITC-labeled anti-GFP mouse monoclonal antibody, and with a PE-labeled anti-vWF, or anti-CD31, or anti-CD90, or anti-CD105 to assess the expression of these cell surface markers on EGFP+ cells (red). PE-labeled mouse isotype immunoglobulins were included in parallel to serve as the negative control (black).

Article Snippet: © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from anti-mouse IgG antibody or PE-labeled anti-hvWF; and PE-labeled mouse monoclonal antibodies against human CD14 (PharMingen, San Diego, CA), CD31, CD90, CD105 (Serotec, Raleigh, NC), and smooth muscle actin (Chemicon, Temecula, CA).

Techniques: In Situ, Agarose Gel Electrophoresis, Amplification, Sequencing, Residue, In Vivo, Staining, Labeling, Expressing, Negative Control

Journal: Bone marrow transplantation

Article Title: Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads.

doi: 10.1038/sj.bmt.1705358

Figure Lengend Snippet: Figure 2 Immunophenotype of mesenchymal stem cells isolated by fibrin microbeads (FMB). (a) FACS analysis of the immunophenotype profile for hematopoietic marker CD45 and for mesenchymal antigens CD90 and CD105 of the initial peripheral blood progenitor cell sample before isolation. Cells isolated by FMB technique were harvested 18–19 days following harvesting and cultured for further passages. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (b) Cells from passages 2–3 were harvested and labeled with antibodies against CD45, CD34, CD90, CD105, fibronectin (FN) and vimentin (Vim) and analyzed by FACS. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (c) Cells from passage 5 were harvested and immunostained with antibodies to CD45 (Cy3 labeled), CD34 (Cy3 labeled), CD90 (Cy3 labeled), CD105 (Cy3 labeled), FN (Cy3 labeled) and Vim (Cy2 labeled). The fluorescence images were merged with dim-light images of the same field to show cell contours.

Article Snippet: The cell suspension was then incubated with the following primary antibodies to the different markers : mouse anti-human CD45 (1:10), mouse anti-human CD34 (1:10), mouse anti-human CD90 (1:10), mouse anti-human CD105 (1:10), all from Serotec, Oxford, UK, mouse anti-human fibronectin (1:500) and rabbit antihuman vimentin (1:50) both from Sigma-Aldrich, Rehovot, Israel.

Techniques: Isolation, Marker, Cell Culture, Control, Staining, Labeling