polyclonal sheep anti cd109 antibody  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Investigation of the E1 and B3 nanobodies' interaction with native CD109 using biolayer interferometry. A and B , the interaction between Nb E1 or B3 and CD109 was investigated using biolayer interferometry (BLI). Nb E1 or B3 were immobilized onto biosensors using amine-reactive chemistry and incubated with native soluble CD109 at concentrations from 12.5 to 200 nM. The experimental curves ( solid lines ) are shown with a baseline reference subtracted (from an Nb-coated biosensor kept in buffer without CD109). Nonspecific binding of CD109 to the blank AR2G biosensors is shown in A and was at most 0.05 nm, showing that most of the response in this experiment was specific. Fitted curves ( dotted lines ) were obtained by the approach given in and all fitted constants are given in and .

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Bivalent binding of immobilized CD109 by E1-Fc and B3-Fc enhances their functional affinity. A and B , the interaction between immobilized E1-Fc or B3-Fc and soluble CD109 was investigated using biolayer interferometry (BLI). E1-Fc ( A ) and B3-Fc ( B ) were immobilized onto anti-human Fc capture (AHC) biosensors and incubated with native CD109 at concentrations from 12.5 to 200 nM for E1-Fc or 12.5 to 800 nM for B3-Fc. The experimental curves ( solid lines) are shown with a baseline reference subtracted (from an Nb-Fc–loaded biosensor kept in buffer without CD109). Nonspecific binding of CD109 to the blank AHC biosensors is shown in B and was at most 0.15 nm, showing that most of the response in this experiment was specific. C and D , the interaction between native CD109 immobilized on a biosensor to soluble E1-Fc and B3-Fc was investigated using BLI. CD109 (10 μg/ml) was immobilized onto AR2G biosensors and subsequently incubated with E1-Fc ( C ) or B3-Fc ( D ) at concentration from 25 to 100 nM. Nonspecific binding of Nb-Fc's to blank AR2G biosensors is shown in C and was at most 0.05 nm. A – D , fitted curves (dotted lines) were obtained by the approach given in and all fitted constants are given in , , , and .

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Binding Assay, Functional Assay, Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: The E1 and B3 nanobodies co-elute with CD109 during size-exclusion chromatography. A , the binding of Nb E1 to CD109 in its native conformation and Nb B3 to both native CD109 and cleaved CD109 (CD109-TEV) was analyzed using SEC. CD109 and nanobodies were incubated together for 30 min at room temperature before loading onto the SEC column. CD109:Nb E1 eluted earlier than CD109 alone, whereas CD109:Nb B3 and CD109-TEV:Nb B3 eluted at the same volume as CD109 and CD109-TEV alone, respectively. B , SDS-PAGE analysis of the fraction containing most protein from each SEC peak confirmed that Nb E1 co-eluted with CD109, while Nb B3 co-eluted with both CD109 and CD109-TEV. C , the binding of E1-Fc and B3-Fc to CD109 was analyzed using SEC. CD109 and Nb-Fc's were incubated for 30 min at room temperature before SEC analysis. CD109 pre-incubated with Nb-Fc's eluted earlier than CD109 alone. The CD109:E1-Fc complex eluted as two peaks, suggesting 1:2 (Nb-Fc:CD109) and 1:1 complex formation, respectively. In contrast, the CD109:B3-Fc complex primarily eluted as a single peak, indicating predominant 1:1 complex formation. In conclusion, E1 and B3, in both their Nb and Nb-Fc fusion forms, bind CD109 and remain associated during SEC.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Size-exclusion Chromatography, Binding Assay, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1 and B3 antagonize CD109's interaction with proteases. A and B , reducing SDS-PAGE showing the effect of E1 ( A ) B3 ( B ) and a negative control nanobody ( C ) on the proteolytic cleavage of CD109. CD109 was pre-incubated with a 1:0.5 to 1:3 CD109:Nb molar ratio for 30 min at room temperature and digested with Cy5-labeled chymotrypsin (CT) for 15 min at 37 °C. Chymotrypsin was inhibited with 2 mM PMSF for 15 min at room temperature before denaturation and reduction. The observed MW variations of the Cy5-chymotrypsin-CD109 conjugation bands reflect the autolytic digestion of chymotrypsin into peptides of different sizes, which are separated upon denaturation. The Cy5-fluorescent image of the gel, shown below the Coomassie-stained gel, shows that when CD109 is incubated with chymotrypsin without nanobodies present, Cy5-labeled chymotrypsin is detected in high molecular weight (MW) bands corresponding to CD109-chymotrypsin conjugation products. A , the addition of E1 results in a decrease in the abundance of conjugation products. B , B3 prevents both CD109 cleavage (as determined by Coomassie staining) and CD109's conjugation of chymotrypsin (as determined by Cy5-fluorescence). C , the negative control nanobody does not affect cleavage or protease conjugation. D and E , to determine whether E1 increased the extent of CD109 cleavage by chymotrypsin, CD109 was pre-incubated with a 1:2 CD109:Nb molar ratio of E1 ( D ) or negative control nanobody ( E ), then cleaved with a serial titration of chymotrypsin (from 0.0125 to 0.4 M ratios of chymotrypsin to CD109) for 60 min at 37 °C, and finally inhibited with 2 mM PMSF before analysis by reducing SDS-PAGE. The Cy5-fluorescence images show that E1 decreased conjugation of chymotrypsin by CD109, and the Coomassie-stained images show that CD109 is approximately 8-fold more readily cleaved when bound by E1, consistent with prevention of its chymotrypsin inhibition by E1.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: SDS Page, Negative Control, Incubation, Labeling, Conjugation Assay, Staining, High Molecular Weight, Fluorescence, Titration, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1 and B3 epitopes identified by negative stain electron microscopy. A , 3D reconstructions of CD109 ( center ), CD109:E1 ( middle ), and ( right ) CD109:B3 obtained via negative stain electron microscopy (nsTEM). Examples of the exposure images and selected 2D classes from which these are derived are given in A . B , the native CD109 structure (PDB ID 8S3O , colored as in A ) was fitted into the nsTEM-derived 3D reconstructions. Additional densities were observed for CD109:E1 and CD109:B3. Predicted models were generated using AlphaFold3 for the E1 nanobody interacting with the TE domain and the B3 nanobody interacting with the MG4 domain as shown in . These models are grafted onto native CD109 (with the nanobody colored red ), showing a single representative model for E1 and five models for B3 which showed more variation in its AlphaFold3-derived output models. C and D , AlphaFold3-derived models showing E1 ( red , CDRs in yellow ) interaction with the TE domain ( blue ) and B3 ( red , CDRs in yellow ) interacting with the MG4 domain ( green ).

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Staining, Electron Microscopy, Derivative Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: CD109 structure and function. A , the domain organization of CD109. The bait region (BR) sequence with identified cleavage sites ( , , ), the thiol ester ( yellow circle , TE), the furin cleavage site, and the GPI anchor are highlighted. B , a schematic illustration of the protease-inhibitory mechanism of CD109. A protease cleaves the bait region of CD109, triggering a conformational change. During the conformational change, a previously hidden thiol ester is exposed, allowing it to conjugate the protease. This structural rearrangement disrupts noncovalent interactions between the MG8 domain and the rest of CD109, leading to the release of CD109 from the cell surface. C and D , cartoon representation of native and activated CD109 structures determined by cryo-EM (PDB accession codes 8S3O and 9FX3). Domain colors correspond to those in ( A ). The position of the thiol ester is indicated in both structures. The bait region is not modeled in native CD109 but the 55 Å distance that it spans between Asp649 and His689, indicated with a dotted red line . The cavity that is occupied by trapped proteases is indicated by an orange circle in cleaved CD109. Note that in these structures, CD109 is oriented with its TE domain and MG8 domain facing upwards, which is upside-down compared to its orientation in ( B ).

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Sequencing, Cryo-EM Sample Prep

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Schematic illustrations of the E1 and B3 mechanisms of action. A , the protease-inhibitory mechanism of CD109. A protease cleaves the bait region of CD109, triggering a conformational change. During the conformational change, a previously hidden thiol ester is exposed, allowing it to conjugate the protease. This structural rearrangement disrupts noncovalent interactions between the MG8 domain and the rest of CD109, leading to the release of CD109 from the cell surface. B , E1's mechanism of action. E1 binds to the TE domain. When a protease cleaves the bait region, E1 temporarily stabilizes the native conformation, slowing down the conformational change. This delay allows the protease to diffuse away, favoring hydrolysis of the thiol ester over conjugation of proteases. C , B3's mechanism of action. Nb B3 binds in the vicinity of the bait region and sterically hinders proteases from accessing the bait region and cleaving CD109.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Conjugation Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1-Fc and B3-Fc antagonize CD109's inhibition of chymotrypsin. A , CD109 was incubated with E1-Fc or B3-Fc in a (1:1) or (1:2), respectively, molar ratio of CD109:Nb-Fc, or buffer only for 30 min at room temperature. Chymotrypsin (#C3142, Sigma-aldrich) was added to the indicated ratios of CD109 in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl2, pH 8 for 15 min, after which DQ-labeled gelatin was added as a chymotrypsin substrate. The fluorescence of digested gelatin was measured after 20 min. Chymotrypsin activity is normalized to its activity without the addition of CD109 or Nb-Fc. B , chymotrypsin activity at a 0:1 and 32:1 CD109:chymotrypsin molar ratio, with or without the addition of Nb-Fc. The significance of the inhibition of chymotrypsin by CD109 and the antagonism of CD109's inhibition by E1-Fc and B3-Fc was tested by unpaired two-tailed t-tests, and the resulting p -values are shown on the diagram. Chymotrypsin was significantly inhibited by CD109, and E1-Fc and B3-Fc significantly antagonized this inhibition. Both Fc constructs increased the activity of chymotrypsin when added without CD109 (approximately 20% increase), likely due to stabilizing chymotrypsin before gelatin addition. This stabilization is also conveyed by CD109 and can therefore be neglected when comparing the effect of CD109 with CD109-antibody complexes. Data are shown as the mean values ± SD, n = 3 (technical replicates). C , the ability of E1-Fc and B3-Fc to prevent CD109's chymotrypsin inhibition was investigated using α2-macroglobulin (A2M) as the protease substrate. CD109 was incubated with B3-Fc at a 1:2 M ratio of CD109: B3-Fc or with E1-Fc at a 1:1 M ratio for 15 min. Chymotrypsin was then added to a 1:18 protease:CD109 M ratio for 10 min at 37 °C. Then, A2M was added (using an amount of A2M giving a 1.2:1 chymotrypsin:A2M M ratio) and digested for an additional 5 min at 37 °C. Chymotrypsin was then inhibited with 2 mM PMSF prior to PAGE analysis. Controls were included which added buffer instead of CD109, antibody, and/or chymotrypsin but otherwise kept conditions identical. The samples were then analyzed by pore-limited native PAGE to determine the conformation of A2M. CD109 inhibited chymotrypsin's cleavage of A2M, and E1-Fc and B3-Fc antagonized this inhibition allowing chymotrypsin to cleave A2M despite pre-incubation with CD109.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Inhibition, Incubation, Labeling, Fluorescence, Activity Assay, Two Tailed Test, Construct, Clear Native PAGE

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: B3-Fc blocked the release of CD109 from the cell surface. A , HEK293F cells were transfected with membrane-bound CD109. Three hours post-transfection, cells were treated with 50 nM Nb-Fc's or media alone. This treatment was administered four times in total, and cells were harvested on day 4. B , cell media was then analyzed by Western blotting with a polyclonal anti-CD109 antibody (R&D Systems, #AF4385). The Western blot results show that when cells were treated with B3-Fc, N- and C-terminal fragments were not present in the media, whereas they were detected in cells treated with E1-Fc or media alone. This suggests that B3-Fc prevents the protease-mediated release of CD109 from the cell surface. Note that the human Fc regions of the Nb-Fc fusions were nonspecifically bound by the anti-sheep antibody-HRP conjugate used for detection. C , a schematic illustration of B3 and E1 mechanisms of action on cells. B3-Fc binds near the bait region of membrane-bound CD109 and blocks protease cleavage, preventing the protease-mediated release of CD109 from the cell surface. E1-Fc binds to the TE domain on CD109 and when an incoming protease cleaves the bait region, E1-Fc prevents conjugation of the protease and thus the protease remains active. E1-Fc dissociates from CD109 after CD109 is cleaved.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Transfection, Membrane, Western Blot, Conjugation Assay

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: Correlation of CD109 protein expression with tumor grade, p-STAT3, and Ki-67 in diffusively infiltrating astrocytomas (grades II–IV) or in glioblastomas alone (grade IV)

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: 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: 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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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: Correlation of CD109 protein expression with tumor grade, p-STAT3, and Ki-67 in diffusively infiltrating astrocytomas (grades II–IV) or in glioblastomas alone (grade IV)

Article Snippet: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

Techniques: 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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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: Immunofluorescence: polyclonal sheep CD109 (AF4385, R&D Systems, Bio-Techne); monoclonal rabbit cleaved caspase-3 (Asp175) (9664, Cell Signaling Technology); phalloidin-TRITC, F-actin (P1951, MilliporeSigma); monoclonal mouse anti-human vimentin Cy3 conjugate (C9080, MilliporeSigma); monoclonal rat anti-mouse podocalyxin (MAB1556, R&D Systems, Bio-Techne) and CD31 (553370, BD Biosciences); monoclonal mouse anti-human nuclear marker (NUMA) Cy3 conjugate (MAB1281C3, MilliporeSigma); polyclonal rabbit NG2 (AB5320, MilliporeSigma) and anti-mouse collagen type IV (AB756P, MilliporeSigma); monoclonal mouse α-SMA Cy3 conjugate (C6198, MilliporeSigma); monoclonal rabbit anti-human lamin A/C (ab108595, Abcam); polyclonal goat IBA-1 (ab107159, Abcam); polyclonal goat anti-human PDGFR-α (AF-307 R&D Systems, Bio-Techne); polyclonal rabbit anti-mouse PDGFR-β (sc-436, Santa Cruz Biotechnology).

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