Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Representative micrographs of negative, low, moderate, and high CD109 IHC staining of clinical glioma samples. Scale bar: 50 μm. ( B ) Association of CD109 expression with tumor grade ( n = 346). P < 0.007, χ 2 test. See also . ( C ) Kaplan-Meier survival analysis based on CD109 expression. The median value was used as cutoff; CD109 hi ( n = 118), red line, and CD109 lo ( n = 122), black line. P = 0.024, log-rank test. ( D and E ) Association of CD109 expression with p-STAT3 ( n = 78; P = 0.002) ( D ) and Ki-67 ( n = 173; P = 0.041) ( E ) in glioblastomas, χ 2 test. See also . ( F ) CD109 mRNA expression between glioblastoma ( n = 489) and nontumor specimens ( n = 10) in the TCGA glioblastoma (TCGA GBM) data set. P < 0.001, Tukey’s post hoc test. ( G ) Heatmap shows CD109 mRNA expression between different glioblastoma subtypes across several glioblastoma data sets: mesenchymal (MES), classical (CL), and proneural (PN). ( H ) Heatmap shows mRNA expression levels of CD109 , IL6ST , STAT3 , and IL-6 between glioblastoma cell lines across different subtypes in the HGCC data set.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Immunohistochemistry, Expressing

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Heatmap shows the classification of patient-derived GSCs and the H2 cell line based on their dominant transcriptional subtype: mesenchymal-like (MES), classical-like (CL), and proneural-like (PN). ( B and C ) Western blot analyses of CD109, SOX2, and OLIG2 expression in GSCs and differentiated glioma cells cultured in FBS-containing medium at the indicated time points. ( D ) Western blot analysis of CD109 expression and p-STAT3 levels in GSCs of different glioblastoma subtypes. ( E and F ) Western blot analyses of SOX2 and OLIG2 expression in CD109-silenced GSCs and nontargeted controls at the indicated time points. ( G and H ) Real-time quantitative PCR (qRT-PCR) analysis of GSC marker mRNA levels in CD109-silenced and nontargeted control GSCs at day 11. ( I ) Cell proliferation of CD109-silenced and nontargeted control GSCs at indicated time points. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; **** P < 0.0001, 2-way ANOVA with Tukey’s multiple comparisons test. ( J ) Representative micrographs of colony formation in methylcellulose after CD109 silencing. Scale bar: 200 μm. ( K and L ) Quantification of colonies in methylcellulose. Data are presented as mean ± SD. **** P < 0.0001, 1-way ANOVA with Dunnett’s multiple comparisons test. ( M ) Representative micrographs of GSC growth in 3D fibrin matrix after CD109 silencing. Scale bar: 200 μm. Inset: original magnification, ×10. See also . ( N ) Cell viability in 3D fibrin matrix at day 15. Data are presented as mean ± SD. **** P < 0.0001, unpaired 2-tailed t test. See also . ( O ) Western blot analysis of p-STAT3 levels after CD109 silencing. Data are from n = 2 ( B and C ) and n = 3 ( D – O ) independent experiments. Representative Western blots are shown where β-tubulin served as a loading control.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Derivative Assay, Western Blot, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Control

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) qRT-PCR analysis of IL6R and IL6ST mRNA levels in GSCs. Data are presented as mean ± SD. ( B and C ) Western blot analyses of p-STAT3 levels in CD109-silenced and nontargeted control GSCs after stimulation with IL-6 cytokine (50 ng/mL) for 15 and 30 minutes. ( D ) qRT-PCR analysis of IL6ST mRNA levels in CD109-silenced and nontargeted control GSCs. Data are presented as mean ± SEM. * P < 0.05; **** P < 0.0001, unpaired 2-tailed t test. ( E ) Western blot analysis of GP130 expression after CD109 silencing in GSCs of different glioblastoma subtypes. ( F and G ) Western blot analyses of CD109 expression and p-STAT3 levels in GP130-silenced and nontargeted control GSCs. ( H ) Co-IP analysis of CD109. GSC whole-cell extracts were subjected to immunoprecipitation with an anti-GP130 antibody or appropriate IgG control followed by Western blotting with an anti-CD109 antibody. Inputs are indicated. ( I and J ) Representative micrographs of PLA in GSCs using anti-CD109 and anti-GP130 antibodies. Appropriate IgG controls served as negative controls. Red indicates specific interaction signal. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. ( K and L ) Quantification of PLA signal per cell (DAPI). Data are presented as mean ± SD. ** P < 0.01; **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test. Data are from n = 3 ( A – G ) and n = 2 ( H – L ) independent experiments. Representative Western blots are shown, where β-tubulin served as a loading control.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Quantitative RT-PCR, Western Blot, Control, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A and B ) qRT-PCR analysis of MAP2 , GALC , and GFAP mRNA levels in CD109-silenced and nontargeted GSCs at day 11. Data are presented as mean ± SEM. ** P < 0.01; **** P < 0.0001, 2-way ANOVA with Dunnett’s multiple comparisons test. ( C ) Heatmap shows increased expression of AC-like genes after CD109 silencing at day 11. Data are presented as fold change relative to control. ( D and E ) qRT-PCR analysis of CL subtype genes in CD109-silenced and nontargeted GSCs at d11. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, unpaired 2-tailed t test. ( F ) Cell viability of CD109-silenced and nontargeted GSCs at the indicated time points. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01, 1-way ANOVA with Kruskal-Wallis post hoc test. ( G and H ) Immunofluorescence staining of cleaved caspase-3 (green) and F-actin (red) in CD109-silenced and nontargeting control GSCs in 3D fibrin matrix at day 15. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. ( I ) Box-and-whisker plot shows cell viability of CD109-silenced and nontargeting control GSCs after treatment with 250 μM of TMZ for 4 days. Data were normalized to the corresponding vehicle control. **** P < 0.0001, nonparametric Mann-Whitney U test (BT12) and unpaired 2-tailed t test (BT13 and S24). ( J ) Box-and-whisker plot shows cell viability after treatment with Stattic or combination of Stattic and TMZ. Data were normalized to the corresponding vehicle control. * P < 0.05; ** P < 0.01, nonparametric Mann-Whitney U test. Box plots represent the first quartile, median, and third quartile, with whiskers indicating minimum and maximum values. Data are from n = 3 ( A , B , and D – J ) independent experiments.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Quantitative RT-PCR, Expressing, Control, Immunofluorescence, Staining, Whisker Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: Representative micrographs of coronal sections of tumor-bearing animal brains after implantation of nontargeted control or CD109-silenced BT12 ( A ), BT13 ( B ), BT18 ( C ), or ZH305 ( D ) GSCs. Frozen sections were immunostained with anti-human vimentin (green). Nuclei were counterstained with DAPI (white). Scale bar: 2 mm. Quantification of the BT12 ( E ) and BT13 ( F ) tumor volume, number of BT12 ( G ) and BT13 ( H ) satellite tumors, and number of single invasive BT12 ( I ) and BT13 ( J ) cells of control and CD109-silenced BT12 ( n = 10) and BT13 ( n = 10) xenografts. Quantification of the BT18 ( K ) and ZH305 ( L ) tumor area and number of single invasive BT18 ( M ) and ZH305 ( N ) cells of control and CD109-silenced BT18 ( n = 7) and ZH305 ( n = 6) xenografts. Quantification of the number of Ki-67 + BT12 ( O ) and BT13 ( P ) tumor cells and number or OLIG2 + BT12 ( Q ) and BT13 ( R ) tumor cells of control and CD109-silenced BT12 ( n = 10) and BT13 ( n = 10) xenografts. Western blot analysis of expression of indicated proteins from the whole brain section extracts of BT12 ( S ) and BT13 ( T ) control and CD109-silenced BT12 ( n = 3) and BT13 ( n = 3) xenografts. ( U ) Representative micrographs of BT12 xenografts immunostained with anti–PDGFR-α (white), anti-human vimentin (green), and podocalyxin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. ( V ) Representative micrographs of BT12 xenografts immunostained with anti-NG2 (red, arrows) and anti-human nuclear marker (NUMA, green). Nuclei were counterstained with DAPI (blue). Scale bars: 1 mm or 25 μm as indicated. ( W ) Quantification of the number of PDGFR-α + cells in control and CD109-silenced BT12 ( n = 10) xenografts. ( X ) Quantification of the number of NG2 + cells in control and CD109-silenced BT12 ( n = 10) xenografts. P values were calculated using unpaired 2-tailed t test.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Control, Western Blot, Expressing, Marker

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Representative micrographs of BT12 xenografts immunostained with anti–collagen IV (green) and anti-podocalyxin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. Arrows indicate empty basement membrane sleeves devoid of endothelium. ( B ) Quantification of collagen IV + /PODXL – empty sleeves in control and CD109-silenced BT12 xenografts ( n = 10). ( C ) Representative micrographs of BT12 xenografts immunostained with anti-human vimentin (green) and anti-mouse CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 μm as indicated. Quantification of the average tumor blood vessel size ( D ) and density ( E ) in control and CD109-silenced BT12 xenografts ( n = 10). ( F ) Quantification of the secreted ANG1 and ANG2 levels from the cell culture supernatants of CD109-silenced and nontargeted control BT12 GSCs ( n = 2). ( G ) Expression of ANGPT1 and ANGPT2 mRNA levels determined by total RNA-Seq in CD109-silenced and nontargeted BT12 GSCs at the indicated time points. ( H ) Representative micrographs of BT12 xenografts immunostained with anti-human NUMA (green) and anti-NG2 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 μm as indicated. ( I ) Representative micrographs of BT12 xenografts immunostained with anti–α-SMA (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. ( J ) Quantification of the α-SMA + tumor pericytes in control and CD109-silenced BT12 xenografts ( n = 10). ( K ) Representative micrographs of BT12 xenografts immunostained with anti-NG2 (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. ( L ) Quantification of the pericyte coverage in control and CD109-silenced BT12 xenografts ( n = 10). P values were calculated by using the unpaired 2-tailed t test except for F and G , where a t test with Mann-Whitney post hoc test was used.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Membrane, Control, Cell Culture, Expressing, RNA Sequencing, MANN-WHITNEY

Journal: Oncotarget

Article Title: Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

doi: 10.18632/oncotarget.25017

Figure Lengend Snippet: (A) Immunoprecipitation was performed with novel mAbs KU42.33C and KU43.13A using sheep anti-mouse dynabeads. A protein band of ∼170 KDa was immunoprecipitated with both novel mAbs. The ∼50/25 KDa bands represent heavy and light chains of the anti-mouse antibody, respectively. (B) CD109 antigen was immunoprecipitated and probed with novel mAbs KU42.33C and KU43.13A. CD109 was immunodetected by mAb KU42.33C but not by mAb KU43.13A. (C) CD109 antigen was immunoprecipitated with mAbs KU42.33C and KU43.13A and subsequently immunodetected with commercial anti-CD109 mAb. MWM: molecular weight marker.

Article Snippet: The transfer of proteins from 4-12% Bis-Tris-gels to Immobilon-FL PVDF membranes (Merck Millipore, UK) was performed using the XCell IITM Mini-Cell Blot Module kit (Invitrogen, UK) at a constant voltage of 30 V on ice for 2 h. PVDF membranes were probed with novel mAbs (30 μg/ml) or commercial anti-CD109 mAb (1:100; sc-365780; Santa Cruz Biotechnology, UK) overnight at 4°C and subsequently incubated with secondary goat anti-mouse antibody (1:10,000; LI-COR Biosciences, UK) for 1 h at room temperature.

Techniques: Immunoprecipitation, Molecular Weight, Marker

Journal: Oncotarget

Article Title: Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

doi: 10.18632/oncotarget.25017

Figure Lengend Snippet: Identification of proteins recognised by novel mAbs KU42.33C and KU43.13A by mass spectrometry

Article Snippet: The transfer of proteins from 4-12% Bis-Tris-gels to Immobilon-FL PVDF membranes (Merck Millipore, UK) was performed using the XCell IITM Mini-Cell Blot Module kit (Invitrogen, UK) at a constant voltage of 30 V on ice for 2 h. PVDF membranes were probed with novel mAbs (30 μg/ml) or commercial anti-CD109 mAb (1:100; sc-365780; Santa Cruz Biotechnology, UK) overnight at 4°C and subsequently incubated with secondary goat anti-mouse antibody (1:10,000; LI-COR Biosciences, UK) for 1 h at room temperature.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: Patient information

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Modification

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: CD109 protein expression is increased in systemic sclerosis skin and in cultured systemic sclerosis skin fibroblasts . (A, B) Immunohistochemistry: systemic sclerosis (SSc) (Patients S1 and S19 in Table 1) and normal (controls N1 and N9) skin sections were analyzed by immunohistochemistry using an anti-CD109 antibody followed by detection with (A) Alexa Fluor 488-conjugated secondary antibody (Patient S1 and control N1) or (B) horseradish peroxidase-conjugated secondary antibody (Patient S19 and control N9) followed by diaminobenzidine staining, as described in Materials and methods. (C) Immunocytochemistry: SSc (Patient S1) and normal (control N1) skin fibroblasts were grown in culture and were analyzed by immunocytochemistry using an anti-CD109 antibody followed by detection with an Alexa Fluor 488-conjugated secondary antibody, as described in Materials and methods. Immunofluorescence microscopy was done using Olympus B202 (Carson Group Inc., Markham, ON, Canada). Images captured with a digital camera (DC-330; Dage-MTI Inc., Michigan City, IN, USA). Scale bar: 50 μM. Results presented are representative of three independent experiments. (D, E) Western blot: cell lysates prepared from cultured SSc and normal keratinocytes were analyzed by western blot using an anti-CD109 antibody. The membrane was reprobed with an anti-β-actin antibody to confirm equal protein loading (D). Densitometric analysis of CD109 protein levels (180 kDa band) in the cell lysates of SSc ( n = 6) and normal ( n = 6) keratinocytes was performed using β-actin as a loading control ( P < 0.05).

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Expressing, Cell Culture, Immunohistochemistry, Control, Staining, Immunocytochemistry, Immunofluorescence, Microscopy, Western Blot, Membrane

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: CD109 protein, but not mRNA, expression is increased in systemic sclerosis skin fibroblasts . (A) Cell lysates prepared from systemic sclerosis (SSc) (in = 6, Patients S1 to S6) and normal ( n = 4, controls N1 to N4) skin fibroblasts were analyzed by western blot using an anti-CD109 antibody, as described in Materials and methods. The membrane was reprobed with an anti-β-actin antibody to confirm that equal amounts of protein were loaded in each lane. (B) Densitometric analysis of CD109 protein levels (180 kDa band) in the cell lysates of SSc and normal skin fibroblasts was performed using β-actin as a loading control. Results from two independent experiments corresponding to skin fibroblasts from two different randomly selected groups of SSC patients versus normal subjects are shown: Experiment 1, normal ( n = 4, controls N1 to N4) and SSc ( n = 7, Patients S1 to S7); Experiment 2, normal ( n = 5, controls N5 to N9) and SSc ( n = 9, Patients S8 to S16). P values denote statistical differences of the means between normal and SSc fibroblasts using Student's t test. (C) Data presented in (B) obtained from 16 SSc patients (nine with limited SSc and seven with diffuse SSc) and nine normal healthy controls were reorganized into normal control, limited SSc and diffuse SSc groups and were reanalyzed. One data point (outlier) from the control group (value = 1.04) was removed. Fold-change in the ratio of CD109/β actin in the limited SSc and diffuse SSc groups compared with normal controls is shown. One-way analysis of variance analysis showed a statistically significant difference ( P = 0.0127); Holm-Sidak analysis overall significance level = 0.05. Significant differences observed are indicated. (D) Total RNA extracted from SSc ( n = 7, Patients S1, S6, S8, S14, S7, S12 and S5) and normal ( n = 7, controls N1 to N7) skin fibroblasts was analyzed by RT-PCR using human CD109-specific (top panel) or GAPDH-specific (bottom panel) oligonucleotide primers. PCR products were resolved by agarose gel (1.5%) electrophoresis and visualized by ethidium bromide staining. (E) Densitometric analysis of data presented in (D) expressed as the ratio of CD109 PCR product to GAPDH internal control (CD109/GAPDH) is shown ( P = 0.717).

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Expressing, Western Blot, Membrane, Control, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Staining

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: CD109 protein expression is not regulated by transforming growth factor beta in systemic sclerosis and normal fibroblasts . Systemic sclerosis (SSc) and normal skin fibroblasts were treated for 24 hours with 0, 5, 10, 25, 50 and 100 pM transforming growth factor beta 1(TGF-β 1 ) Cell lysates were prepared and analyzed by western blot using anti-CD109 (top panel) and anti-fibronectin (middle panel) antibodies. The membrane was reprobed with an anti-β-actin antibody (bottom panel) to confirm that equal amounts of protein were loaded in each lane. Results presented are representative of three independent experiments. The representative SSc fibroblast data shown correspond to SSc Patient S2.

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Expressing, Western Blot, Membrane

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: CD109 inhibits extracellular matrix protein production and Smad2/3 phosphorylation in systemic sclerosis and normal fibroblasts . (A, C) Systemic sclerosis (SSc) skin fibroblasts and (B, D) normal skin fibroblasts were transiently transfected with CD109-specific siRNA or control siRNA. Cell lysates were analyzed by western blot using (A, B) anti-CD109 (top panel), anti-fibronectin (second panel), anti-type I collagen (third panel) or anti-CCN2 (fourth panel) antibodies or (C, D) anti-phosphoSmad2, anti-phosphoSmad3, anti-Smad2 or anti-Smad3 antibodies. Membranes were reprobed with an anti-β-actin (bottom panel) antibody to confirm that equal amounts of protein were loaded in each lane. Results presented are representative of three independent experiments. The representative SSc fibroblast data shown correspond to (A) SSc Patient S2 and (C) SSc Patient S16.

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Phospho-proteomics, Transfection, Control, Western Blot

Journal: Arthritis Research & Therapy

Article Title: CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

doi: 10.1186/ar3877

Figure Lengend Snippet: Recombinant soluble CD109 protein inhibits transforming growth factor beta 1-induced extracellular matrix and CCN2 production . (A) Systemic sclerosis (SSc) and normal skin fibroblasts were left untreated (-) or treated (+) for 24 hours with 100 pM transforming growth factor beta 1 (TGF-β 1 ) in the presence of 1.0 nM recombinant CD109 protein or PBS/0.1% BSA vehicle control. Cell lysates were prepared and analyzed by western blot using anti-fibronectin (top panel), anti-type I collagen (second panel) or anti-CCN2 (third panel) antibodies. Membranes were reprobed with an anti-β-actin (bottom panel) antibody to confirm that equal amounts of protein were loaded in each lane. Results presented are representative of three independent experiments. (B) Densitometric analysis of the data from the above blot was performed using β-actin as a loading control. The representative SSc fibroblast data shown correspond to (A) SSc Patient S21 and (B) SSc Patients S19, S2 and S16.

Article Snippet: Sections were then incubated with a mouse anti-human CD109 antibody (R&D Systems Inc.) overnight at 4°C, followed by an Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (AF-488; Invitrogen) for 2 hours at room temperature.

Techniques: Recombinant, Control, Western Blot

Journal: Clinical immunology (Orlando, Fla.)

Article Title: Comprehensive characterization of pathogenic synovial fluid extracellular vesicles from knee osteoarthritis.

doi: 10.1016/j.clim.2023.109812

Figure Lengend Snippet: Fig. 4. CSPG4, CD109, BGN and NRP1 are the major surface markers of pathogenetic SF EVs in knee OA. Spearman analyses were used to assess correlations between the SF EV surface peptidesSF>PL and the 692 SF EV peptidesrOA-Pos. (A) Heat maps depict the Spearman correlation coefficient r values; significant results were defined by p < 0.05. The majority (643 of 692, 92.9%) of pathogenic SF EV peptidesrOA-Pos were significantly correlated with at least one of the surface peptidesSF>PL, whereas the rest (49 of 692, 7.1%) of SF EV peptidesrOA-Pos were not significantly correlated with any surface peptidesSF>PL. (B) The bar graphs display the per centages of the 692 SF EV peptidesrOA-Pos that were significantly (p < 0.05) positively or negatively correlated with each of the SF EV surface peptideSF>PL.

Article Snippet: As previously reported [17], EV pellets were resuspended in double filtered PBS (df-PBS) filtered by 100 nm filters (EMD Millipore) and stained with fluorescence-conjugated antibodies against human CD163, LRP1 (BD Biosciences), CD109, NRP1 (R&D Systems), CSPG4, MARCO (Novus Biologicals), VSIG4 (ThermoFisher Scientific), PTPRS (LifeSpan) and BGN (Bioss).

Techniques:

Journal: Clinical immunology (Orlando, Fla.)

Article Title: Comprehensive characterization of pathogenic synovial fluid extracellular vesicles from knee osteoarthritis.

doi: 10.1016/j.clim.2023.109812

Figure Lengend Snippet: Fig. 5. Gene expression of SF EV surface markers in knee OA joint tissues. Single cell RNA-seq gene expression data were generated from joint tissues of participants (n = 3) with knee OA undergoing joint replacement surgery, including 11,579 chondrocytes from damaged sites of the medial tibial (lesioned) cartilage (MT), 14,613 chondrocytes from intact sites of the outer lateral tibial (non-lesioned) cartilage (OLT), and 10,640 synoviocytes from the matched synovium (SY) as previously described [12]. (A) The bar graph displays the % of cells expressing genes encoding the surface markers in cells derived from human OA joint specimens of the medial tibia (MT, lesioned articular cartilage), outer lateral tibia (OLT, non-lesioned articular cartilage), and synovium (SY). (B) The violin (density) plots display the frequency of cells expressing each gene in the three types of OA joint tissue cells. (C) The bar graphs display the mean gene expression of the top 10 expressed genes in CSPG4+ and CD109+ MT chondrocytes. (D) The bar graphs display the mean gene expression of the top 10 expressed genes in VISG4+, MARCO+, CD163+, NRP1+ and PTPRS+ synoviocytes. Peptides corresponding to the genes labeled with the “EV” symbol indicate those detected in SF and plasma EVs (Supplementary Table 7), while the peptides corresponding to the genes without the “EV” label were not detected in SF EVs or plasma EVs from knee OA participants (Supplementary Table 2).

Article Snippet: As previously reported [17], EV pellets were resuspended in double filtered PBS (df-PBS) filtered by 100 nm filters (EMD Millipore) and stained with fluorescence-conjugated antibodies against human CD163, LRP1 (BD Biosciences), CD109, NRP1 (R&D Systems), CSPG4, MARCO (Novus Biologicals), VSIG4 (ThermoFisher Scientific), PTPRS (LifeSpan) and BGN (Bioss).

Techniques: Gene Expression, RNA Sequencing, Generated, Expressing, Derivative Assay, Labeling, Clinical Proteomics

Journal: The Journal of Reproduction and Development

Article Title: CD2 is a surface marker for mouse and rat spermatogonial stem cells

doi: 10.1262/jrd.2020-019

Figure Lengend Snippet: CD2 expression in mouse spermatogonial stem cells (SSCs). (A) Flow cytometric analysis of CD109 and CD2 expression in germline stem (GS) cells cultured for 5 months (5M-GS) and GS cells cultured for 60 months (60M-GS cells) (n = 4). Green lines indicate the control. (B) Flow cytometric analysis of wild-type mouse testis cells based on CD109 and CD2 after magnetic cell sorting (MACS) selection with an anti-CDH1 antibody. (C) Colony count (n = 6 for CD109; n = 12 for CD2). (D) Macroscopic appearance of recipient testis transplanted with CD2 high ROSA testis cells. (E) Histological appearance of recipient testis. Bar = 1 mm (D), 50 μm (E). Asterisk indicates statistical significance (P < 0.05). MFI, mean fluorescence intensity.

Article Snippet: For CD109 selection, we used PE-conjugated mouse anti-human CD109 antibody (496920; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Control, FACS, Selection, Fluorescence