Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: GBM CD105 + cell localization and specificity. A Schematic of clinical tissue collection. A total of 19 GBM border tissue samples were collected with guidance from the surgical navigation system. Each sample was split into two parts for different studies. B The landscape of CD105 + cells and cancer cells in GBM border tissue. Brain section containing the border between tumor and normal brain (pre-invasive niche) stained with CD105 showing the distribution of CD105 + cells. The border between tumor and peritumor area is identified by a difference in cell density (yellow line). High magnification images of tumor cells are shown using the markers CD105, SOX2 and Nestin. C Quantification of SOX2 + Nestin + and CD105 + Nestin + cells in tumor and peritumor tissue ** P < 0.01. D Evaluation of CD105 + cells in GBM in vivo models. Sections showing tumors at 25 days following intracranial injection of GL261 and U87 cells in mouse brains. Sections were stained with CD105 and Hu antibodies. Magnified images showing the border between tumor and brain. E Comparing the CD105 expression in 163 GBM samples from TCGA database and 207 normal brain samples from GTEx database * P < 0.05. F Overall survival correlated with CD105 RNA expression as analyzed from the data of 82 GBM patients in the TCGA database

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: Staining, In Vivo, Injection, Expressing, RNA Expression

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: Culture conditions can interfere with the fate of GBM CD105 + cells. A Schematic representation of media conditions for CD105 + primary cell culture. B Primary cell culture in SC and SFC under passage 3 (P3). CD105 + cells and neural stem-like cells were identified by staining of CD105 (red) and SOX2 (green), respectively. C Flow cytometry analysis of CD105 + cells in different media conditions. Flow cytometry data show the GBM CD105 subpopulation obtained from GBM primary cells using the phycoerythrin (PE) channel. *** P < 0.001. D Immunostaining of CD105 + cell with cell type markers. Double immunostaining of CD105 (red) and cellular markers (green) on sorted CD105 + cells (under P3). E , F Effect of serum/ serum-free culture conditions on the differentiation of GBM primary cell lines. SFC cultured SOX2 + cells differentiated into DMEM/F12 media supplemented with 10% FBS for 14 days ( E ). Immunostaining of CD105 (red) and SOX2 (green) shown on differentiated cells (DAPI blue). Bar graph showing cell viability of 5 GBM SOX2 + cell lines differentiated into SC and SFC for 14 days. An exceptional SOX2 + cell line, GBM B17, differentiated in SC with different serum concentrations, showed a higher percentage of CD105 + cells by flow cytometry data. F Sorted CD105 + cells differentiated in SFC supplemented with b-FGF for 14 days. Differentiated cells were stained with CD105 (red) and SOX2 (green). Bar graphs showing quantification of cell viability of (left lower) and CD105 + cell subpopulation (right lower). 5 CD105 + cell lines were differentiated in SFC for 14 days. Graph shows CD105 + cell lines tolerating SFC but losing CD105 marker positivity. * P < 0.05 ** P < 0.01 *** P < 0.001

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: Cell Culture, Staining, Flow Cytometry, Immunostaining, Double Immunostaining, Marker

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: CD105 + cell stemness and tumorigenicity assay. A Schematic of intracranial transplantation for CD105 + cells. Sorted primary CD105 + cell lines (n=5) cultured in 2D and 3D conditions. 250,000 cells were injected into NSG mouse brains. Brain tissue was harvested immediately following the loss of each animal. MSCs cell line from a healthy donor and U87 cell line was used as controls (n=5 in each group). B Sphere formation assay of GBM CD105 + cells. CD105 + cells were cultured in 3D conditions for 14 days and tumorspheres captured under the brightfield or fluorescence microscopy. IF double staining shows CD105 double stained together with SOX2, Nestin and Ki67. C Comparison of the cell viability of CD105 + cells between 2D and 3D cultured conditions. 1000 of 2D or 3D CD105 + cells cultured for 24h. Cell viability assay based on PrestoBlue fluorescence intensity of each CD105 + cell line. *** P < 0.001, * P < 0.05. D Kaplan–Meier survival curves comparing 2D or 3D cultured CD105 + cells xenografts. E Scans of mouse brain tumor sections. F Brain sections of xenografted mice transplanted with 2D or 3D cultured CD105 + cells stained with CD105 (red) and Hu (green) and DAPI (blue)

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: Tumorigenicity Assay, Transplantation Assay, Cell Culture, Injection, Tube Formation Assay, Fluorescence, Microscopy, Double Staining, Staining, Viability Assay

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: Exome sequencing of GBM CD105 + cells. A The number of mutations, annotated genes and the rate of mutant genes matching into the CCLE cancer database were analyzed from the exome sequencing data of 5 GBM CD105 + cell lines. Pie chart quantifying the subtype of mutations. B Circos chart showing the distribution of mutant genes in each chromosome. The area represents the number of mutant genes. C Veen chart displaying common mutant genes among 5 GBM CD105 + cell lines. D Mutant genes matching TCGA and CCLE databases. E The mutant genes of GBM CD105 + cells matching with GBM hallmark genes from COSMIC. Upper panel: Representation of mutation types. Lower panel: Subclone variant allele frequencies (VAF)

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: Sequencing, Mutagenesis, Variant Assay

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: In vitro functional assays of the crosstalk between CD105 + cells and TME. A Angiogenic and immunosuppressive factors detected by antibody assay. POS positive control, NEG negative control, ANG angiogenin, EGF epidermal growth factor, CXCL5 C-X-C Motif Chemokine Ligand 5, FGF-2 fibroblast growth factor-2, GRO growth-related oncogene, IFN-γ interferon gamma, IGF insulin-like growth factor, IL-6 interleukin 6, IL-8 interleukin 8, CCL C-C Motif Chemokine Ligand, PDGF-BB platelet-derived growth factor B-chain homodimer, PLGF placenta growth factor, TGFβ transforming growth factor-β, TIMP tissue inhibitor of metalloproteinase, TPO thyroid peroxidase, VEGF vascular endothelial growth factor. B Heatmap of 20 angiogenic and immunosuppressive factors identified on 6 GBM CD105 + cell lines. Protein expressions are displayed as colors ranging from red to blue as shown in the key. C Quantification of the overexpressed proteins on CD105 + cells (n = 6 cell lines). * P < 0.05, ** P < 0.01, *** P < 0.001. D PD-L1 expression assays on GBM CD105 + cells. Flow cytometry data showing the differential PD-L1 expression on CD105 + cell lines. Lower panels: IF staining verifying the coexpression of CD105 (red) and PD-L1 (green)

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: In Vitro, Functional Assay, Positive Control, Negative Control, Derivative Assay, Expressing, Flow Cytometry, Staining

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: Drug screening in vitro. A , B Cell toxicity assays on CD105 + cells lines. Different concentrations of temozolomide ( A ) and bevacizumab ( B ) on MGMT promoter methylated CD105 + cell line (GBM B16) and MGMT wildtype CD105 + cell line (GBM B14) cultured for 96h. Fluorescence units represent the cell viability detected at 24 h, 48 h, 72 h and 96 h. Control cells were kept in culture medium without adding any drug. C Heatmap showing the toxicity of 88 clinical compounds (10μM) against 3 GBM CD105 + cell lines and U87 cell line. Cell toxicities are displayed by the range of the colors from red to blue as high to low. The rows are clustered using correlation distance. D Cell toxicity assay showing the effect of different concentrations of Doxorubicin, Idarubicin HCl, Fludara and ABT-751 on CD105 + and U87 cells. Cell viability was detected at 48 h and 96 h. ** P < 0.01. E Interaction plot between drugs and genes. Each circle represents a gene, and its area symbolizes relevance. F Kaplan–Meier curves showing the relation between patient survival and drug-interacted genes as analyzed using patient data from TCGA database. P value is calculated by logrank test. * P < 0.05

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: In Vitro, Methylation, Cell Culture, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Glioblastoma CD105 + cells define a SOX2 − cancer stem cell-like subpopulation in the pre-invasive niche

doi: 10.1186/s40478-022-01422-8

Figure Lengend Snippet: Schematic illustrating multiple roles of CD105 + cells in GBM. A CD105 + cell interacts with the TGF-β pathway. CD105, a receptor of TGF-β, can affect angiogenesis, vascular permeability and cell proliferation by binding different TGF-β subtypes. B , C CD105 + cell shapes the tumor immunosuppressive microenvironment. Cytokines secreted by GBM CD105 + cells share the function of recruiting immunosuppressive cells. CD105 + cells impair immune cell function and contribute to creating an immunosuppressive barrier. D CD105 constitute a marker of GBM vasculature. E In vitro drug sensitivity screening of CD105 + cells. CD105 + cells are resistant to conventional drugs: TMZ and AVZ but sensitive to ABT-751, Doxorubicin, Idarubicin and Fludarabine. F Schematic depicts the “Seed and soil” rational for GBM growth. Based on the distribution and function of CD105 + cells, we predict CD105 + cells may release extracellular matrix (ECM), initiating a tumor tolerant microenvironment and thereby promoting tumor progression

Article Snippet: The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz).

Techniques: Permeability, Binding Assay, Cell Function Assay, Marker, In Vitro

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: International Journal of Molecular Sciences

Article Title: Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

doi: 10.3390/ijms22115976

Figure Lengend Snippet: hOMSC characterization. ( A – D ) Immunofluorescence of hOMSCs and pluripotency markers. White arrows mark areas of positivity. ( E ) Flow cytometry gates. Percentage of positive cells in culture for hOMSCs—( F ) CD90 65.8%, ( G) CD73 55.9%, ( H ) CD105 15.4% and pluripotency markers—( I ) Nanog 26.8%, ( J ) Sox2 11.5% and ( K ) Oct3/4, 24.3%.

Article Snippet: They were incubated in a humid chamber with a 1:50 dilution of the primary antibodies—CD105 (goat anti-human), CD90 (goat anti-human); Sox2 (rabbit anti-human), Nanog (rabbit anti-human); Oct3/4 (mouse anti-human), CD73 (mouse anti-human) (Santa Cruz Biotechnology, Dallas, TX, USA), HNA (mouse anti-human) (Abcam PLC, Cambridge, UK) in BSA 1%.

Techniques: Immunofluorescence, Flow Cytometry

Journal: Apoptosis

Article Title: Adipose stromal/stem cells assist fat transplantation reducing necrosis and increasing graft performance

doi: 10.1007/s10495-013-0878-7

Figure Lengend Snippet: Antibodies for rabbit ASC characterization by FACS

Article Snippet: CD105 , Mouse , SN6 , 1/20 , Serotec , No.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Primers

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Calcineurin regulates myofibroblast transformation and TRPC‐6 expression in right ventricular fibroblasts. A, Western blots showing calcineurin, α‐SMA, pSmad3, total Smad3, and GAPDH expression in human right ventricular fibroblasts (RVFB) after stimulation with TGF‐β1 (10 ng/mL for 16 to 24 hours) in the presence and absence of cyclosporine (CS). B and D, mRNA levels of calcineurin, α‐SMA, and TRPC‐6 in human RVFB after stimulation with TGF‐β1 in the presence and absence of CS (n=3/group). E, Western blot showing silencing of TRPC‐6 in human RVFB. F, Western blot showing calcineurin and α‐SMA levels in human RVFB after TGF‐β1 stimulation in the presence and absence of a siRNA against TRPC‐6 (siTRPC‐6). * P <0.05 versus vehicle; † P <0.05 versus TGF‐β1 stimulation; ‡ P <0.05 versus WT+TGF‐β1 stimulation. α‐SMA indicates α‐smooth muscle antigen; TGF‐β1, transforming growth factor beta 1; TRPC‐6, transient receptor protein channel 6.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Transformation Assay, Expressing, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Reduced endoglin activity limits calcineurin expression and myofibroblast transformation in right ventricular fibroblasts. A and B, Western blots showing calcineurin, α‐SMA, pSmad3, and GAPDH levels in fibroblasts from human right (RVFB) and left (LVFB) ventricular fibroblasts after TGF‐β1 stimulation in the presence and absence of increasing concentrations of a neutralizing endoglin antibody (N‐Eng Ab). Quantification of calcineurin and a‐SMA levels in human RVFB and LVFB (n=3/group). C, mRNA levels of calcineurin and α‐SMA in right (RVFB) and left (LVFB) ventricular fibroblasts derived from WT and Eng +/− mice after TGF‐β1 stimulation (n=6/group). D, Western blots showing calcineurin and α‐SMA levels after TGF‐β1 stimulation in RVFB and LVFB from WT and Eng +/− mice. D and E, Quantification of calcineurin and α‐SMA protein levels in RVFB and LVFB from WT and Eng +/− mice stimulated with TGF‐β1. * P <0.05 versus vehicle; † P <0.05 versus TGF‐β1 stimulation; ‡ P <0.05 versus LVFB+TGF‐β1 stimulation. α‐SMA indicates α‐smooth muscle antigen; TGF‐β1, transforming growth factor beta 1; WT, wild type.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Activity Assay, Expressing, Transformation Assay, Western Blot, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Reduced endoglin expression limits fibrosis and calcineurin expression in a murine model of angio‐obliterative pulmonary hypertension. A through C, Right ventricular systolic pressure, tau, and RV compliance in Eng +/+ and Eng +/− mice after 5 weeks of treatment with Sugen compound under normoxic (Su‐Norm) or hypoxic (Su‐Hypox) conditions (n=6/group). D, mRNA levels of type I collagen in WT and Eng +/− mice under Su‐Norm or Su‐Hypox conditions (n=6/group). E and F, Representative histologic staining for RV collagen abundance in Eng +/+ and Eng +/− mice under Su‐Norm or Su‐Hypox conditions. Quantification of percent RV fibrosis is shown (n=6/group). G, mRNA levels of calcineurin, TRPC‐6, and a‐SMA in RV tissue from WT and Eng +/− mice under Su‐Norm or Su‐Hypox conditions (n=6/group). * P <0.05 versus Eng +/+ Su‐Norm; † P <0.05 versus Eng +/− Su‐Norm; ‡ P <0.05 Eng +/+ Su‐Hypox versus Eng +/− Su‐Hypox. α‐SMA indicates α‐smooth muscle antigen; RV, right ventricular; TRPC‐6, transient receptor protein channel 6; WT, wild type.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Expressing, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Reduced endoglin expression limits TGF‐β1 signaling and calcineurin activity in the RV after right ventricular pressure overload. A, Levels of active TGF‐β1 in RV protein lysates from WT and Eng +/− mice (n=6/group). B through D, Quantification of RV type I collagen, pSmad3, and pERK1/2 protein levels in WT and Eng +/− mice after PAC (n=6/group). Representative western blots are shown. E, Levels of RV calcineurin protein in WT and Eng +/− mice after PAC (n=6/group). A representative western blot is shown. F and H, Levels of RV MYH7, TRPC‐6, and α‐SMA mRNA expression in WT and Eng +/− mice after PAC (n=6/group). * P <0.05 versus sham; † P <0.05 versus WT‐PAC. α‐SMA indicates alpha‐smooth muscle antigen; PAC, pulmonary artery constriction; RV, right ventricular; TGF‐β1, transforming growth factor beta 1; TRPC‐6, transient receptor protein channel 6; WT, wild type.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Neutralizing endoglin activity improves survival and limits the development of RV fibrosis after right ventricular pressure overload. A, Kaplan‐Meier's survival curves in WT mice treated with an IgG control Ab or N‐ Eng Ab after PAC (n=18/group). B and C, Representative histological staining for RV collagen abundance in IgG vs. N‐Eng Ab‐treated mice after PAC. Quantification of RV fibrosis after PAC is shown (n=6/group). D, Quantification of cardiomyocyte cross‐sectional area after PAC is shown (n=6/group). E through H, Quantification of RV type I collagen, pSmad3, pERK1/2, and calcineurin protein levels in IgG versus N‐Eng Ab‐treated mice after PAC (n=6/group). Representative western blots are shown. I through K, Levels of RV MYH7, TRPC‐6, and α‐SMA mRNA expression in IgG versus N‐Eng Ab‐treated mice after PAC (n=6/group). * P <0.05 versus sham; † P <0.05 versus WT+N‐Eng Ab PAC. α‐SMA indicates alpha‐smooth muscle antigen; PAC, pulmonary artery constriction; RV, right ventricular; TRPC‐6, transient receptor protein channel 6; WT, wild type.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Activity Assay, Control, Staining, Western Blot, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reducing Endoglin Activity Limits Calcineurin and TRPC‐6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

doi: 10.1161/JAHA.114.000965

Figure Lengend Snippet: Reduced endoglin activity limits TGF‐β1‐induced calcineurin expression and myofibroblast transformation in right ventricular fibroblasts. Postulated mechanism by which endoglin promotes RV fibrosis by facilitating TGF‐β1 signaling in response to pressure overload through canonical and noncanonical pathways, including calcineurin‐mediated myofibroblast transformation in RVFB. In contrast, reduced endoglin activity attenuates TGF‐β1 signaling through canonical, noncanonical, and calcineurin pathways and limits myofibroblast transformation and fibrosis, thereby improving survival. α‐SMA indicates alpha‐smooth muscle antigen; RV, right ventricular; RVBF, right ventricular fibroblasts; TGF‐β1, transforming growth factor‐beta 1; TRPC‐6, transient receptor protein channel 6.

Article Snippet: Polyclonal Abs against mouse endoglin, type I collagen, and α‐SMA were purchased from R&D Systems (BAF1320; Minneapolis, MN), Santa Cruz Biotechnology (SC‐25974; Santa Cruz, CA), and Sigma‐Aldrich (A2547), respectively.

Techniques: Activity Assay, Expressing, Transformation Assay

Journal: The FASEB Journal

Article Title: Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization

doi: 10.1096/fj.09-145508

Figure Lengend Snippet: AC is present in human follicular fluid (FF) and cumulus cells. AC expression was determined by Western blotting in 10 μl of human FF (diluted to 1 μg/μl total protein concentration) (A), and 24 μg of total protein from cumulus cell extracts (B). Western blot analysis was performed using mouse anti-human AC IgG, revealing the human AC precursor (at 55 kDa). Western blot images are representative of ≥3 independent experiments.

Article Snippet: The following antibodies were used: anti-human AC goat polyclonal immunoglobulin G (IgG; T-20, sc-28486; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human AC mouse monoclonal immunoglobulin M (IgM; 612302; BD Bioscience, San Jose, CA, USA), anti-human Lamp1 rat monoclonal IgG2a (sc-19992; Santa Cruz Biotechnology), and anti β-tubulin mouse monoclonal IgG (sc-5274; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Protein Concentration

Journal: The FASEB Journal

Article Title: Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization

doi: 10.1096/fj.09-145508

Figure Lengend Snippet: AC expression in human embryos. A, B) Low AC expression in the blastocele fluid and outer cell mass of a representative low-grade human embryo (A) compared with high AC expression in a high-grade embryo (B), detected using goat IgG against human AC. C–J), Lower AC expression and higher apoptosis (TUNEL) in the outer cell mass of a low-grade embryo compared to the inner cell mass of the same embryo, which had higher AC expression and less apoptosis. AC was detected using goat IgG against human AC (E, I) and TUNEL assay (D, H); DNA labeling by Hoechst (C, G); merged images indicate colocalization (F, J). Scale bars = 10 μm. Data are representative of 3 independent experiments.

Article Snippet: The following antibodies were used: anti-human AC goat polyclonal immunoglobulin G (IgG; T-20, sc-28486; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human AC mouse monoclonal immunoglobulin M (IgM; 612302; BD Bioscience, San Jose, CA, USA), anti-human Lamp1 rat monoclonal IgG2a (sc-19992; Santa Cruz Biotechnology), and anti β-tubulin mouse monoclonal IgG (sc-5274; Santa Cruz Biotechnology).

Techniques: Expressing, TUNEL Assay, DNA Labeling

Journal: Biomaterials

Article Title: Quantum dots-based in situ molecular imaging of dynamic changes of collagen IV during cancer invasion.

doi: 10.1016/j.biomaterials.2013.07.069

Figure Lengend Snippet: Fig. 3. Dynamic changes of tumor microenvironment in well-differentiated tubular adenocarcinoma of GC. (A) shows collagen IV and macrophages in peri-tumor tissue of GC. (B) shows GC nests are surrounded by intact and dense collagen IV almost without degradation and ECM deposition. (C) shows collagen IV aligned with GC nests is degraded at invasion front (Red arrows) with increased irregular collagen IV deposition in ECM. (DeF) show increased deposition of collagen IV in tumor stroma, presenting stiff and rigid. (GeH) show CD105 expression in peri-tumor tissue and GC tissue, respectively. (I) shows double-staining of macrophages and CD105 in GC tissue, in which tumor macrophages aggregate. (JeL) show multiplexed imaging of collagen IV, macrophages and CD105, in which cancer cells, macrophages and tumor neo-vessels constitute “invasion unit” (Red square indicates an “invasion unit”). Scale bar: 50 mm for A, D, E, G, H & J; 20 mm for B, C, F & K; 10 mm for I & L.

Article Snippet: After blocked with 2% bovine serum albumin (BSA), the slides were first incubated with primary antibodies for rabbit anti-human polyclonal antibody against collagen IV (ab6586, Abcam, England, dilution 1/300), mouse anti-human monoclonal antibody against macrophages (MA1-38069, ABR, USA, dilution 1/300) and goat anti-human polyclonal antibody against CD105 (sc-20072, Santa Cruz, USA, dilution 1/500) for 2 h at 37 C, then with corresponding secondary antibody, including peroxidase labeled anti-goat IgG (H þ L) antibody (14-13-06, KPL, USA, dilution 1/300), peroxidase labeled anti-mouse IgG (H þ L) antibody (074-1806, KPL, USA, dilution 1/300) and peroxidase labeled anti-rabbit IgG (Hþ L) antibody (074-1506, KPL, USA, dilution 1/300) for 30 min at 37 C, respectively.

Techniques: Expressing, Double Staining, Imaging

Journal: Journal of Ophthalmology

Article Title: Overexpression of MiR-183/96/182 Triggers Retina-Like Fate in Human Bone Marrow-Derived Mesenchymal Stem Cells (hBMSCs) in Culture

doi: 10.1155/2019/2454362

Figure Lengend Snippet: Flow cytometry analysis of BMSCs: BMSCs (two passage cells) were analyzed via fluorescence-activated cell sorting and Cell Quest software for expression of CD34 (a), CD45 (b), CD73 (c), CD90 (d), and CD105 (e).

Article Snippet: Briefly, the cells were dissociated with trypsin/EDTA; then, cell suspensions were stained using various antibodies against MSC markers including CD105-FITC (R&D Systems), CD90-PE (Dako), CD73-PE (Abcam), CD45-FITC (Dako), and CD34-PE (Biosciences) and secondary goat antimouse IgG-PE.

Techniques: Flow Cytometry, Fluorescence, FACS, Software, Expressing