gpnmb recombinant protein treatment  (R&D Systems)

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) Strategy for identification of the overlapped genes that were upregulated in both GBM-associated microglia and bone marrow–derived macrophage (BMDMs) compared with normal monocytes. The analysis was based on RNA-Seq data ( GSE86573 ) from GBM-associated microglia/BMDMs isolated from the GL261 tumor model and RCAS tumor model and normal microglia and monocytes. ( B ) The expression pattern of genes in CD45 – GBM cells and tumor-associated immune cells (e.g., BMDMs, microglia, neutrophils, CD4 + T cells, and CD8 + T cells) isolated from human GBM tumors based on the Brain TIME dataset . ( C ) Coimmunofluorescence staining for GPNMB (red) and F4/80 (macrophage marker, green) or CX3CR1 (microglia marker, green) in CT2A tumors implanted in C57BL/6 mice. Scale bar: 50 μm. ( D – G ) Flow cytometry analysis of GPNMB expression in CD11b + CD45 hi CD68 + macrophages ( D and E ) isolated from bone marrow and CD11b + CD45 lo CX3CR1 + microglia ( F and G ) isolated from brain tissues of tumor-free C57BL/6 mice and CT2A and QPP7 tumor-bearing C57BL/6 mice. n = 3–6 independent samples. One-way ANOVA test. ( H – K ) Flow cytometry analysis of GPNMB expression in CD11b + CD45 hi CD11c + DCs, CD11b + CD45 lo CX3CR1 + microglia (Total MG), CD11b + CD45 lo CX3CR1 + CD206 + microglia (CD206 + MG), CD11b + CD45 lo CX3CR1 + CD206 – microglia (CD206 – MG), CD11b + CD45 hi CD68 + macrophages (Total MΦ), CD11b + CD45 hi CD68 + CD206 + macrophages (CD206 + MΦ), CD11b + CD45 hi CD68 + CD206 – macrophages (CD206 – MΦ), CD11b + CD68 + Ly6G lo Ly6C hi monocytic immature myeloid cells (M-IMCs), and CD11b + CD68 + Ly6G hi Ly6C lo polymorphonuclear immature myeloid cells (PMN-IMCs) isolated from QPP7 tumors ( H and I ) and CT2A tumors ( J and K ) implanted in C57BL/6 mice. n = 3 independent samples. One-way ANOVA test. ( L ) Immunoblots for GPNMB in lysates of Raw264.7 macrophages and SIM-A9 microglia treated with the conditioned media (CM) of QPP7 GSCs for 24 hours. ( M ) Immunoblots for GPNMB in lysates of THP-1 macrophage and HMC3 microglia treated with the CM of GSC272 cells for 24 hours. ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Derivative Assay, RNA Sequencing, Isolation, Expressing, Staining, Marker, Flow Cytometry, Western Blot

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) GSEA analysis of single-cell RNA-Seq (scRNA-Seq) data from human GBM tumors (EGAS00001004871) shows top enriched WikiPathways signatures in tumors with TAMs expressing high GPNMB compared to low GPNMB . ( B and C ) Extracellular acidification rate (ECAR) of GSC272 cells ( B ) and CT2A cells ( C ) treated with GPNMB recombinant protein (100 ng/mL) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 3 independent samples. ( D and E ) ECAR of GSC272 cells treated with the conditioned media (CM) of THP-1 macrophages ( D ) and HMC3 microglia ( E ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 6 independent samples. ( F – H ) Quantification of relative L-lactate levels in GSC272 ( F ), GSC2 ( G ), and CT2A cells ( H ) treated with GPNMB recombinant protein (100 ng/mL) for 24 hours. n = 3 independent samples. Student’s t test. ( I and J ) Quantification of relative L-lactate levels in GSC272 cells treated with the CM of THP-1 macrophages ( I ) and HMC3 microglia ( J ) expressing shC or sh GPNMB for 24 hours. n = 3 independent samples. One-way ANOVA test. ( K and L ) Quantification of relative L-lactate levels in GSC2 cells treated with the CM of THP-1 macrophages ( K ) and HMC3 microglia ( L ) expressing shC or sh GPNMB for 24 hours. n = 3 independent samples. One-way ANOVA test. ( M and N ) Quantification of relative L-lactate levels in CT2A cells treated with the CM of Raw264.7 macrophages ( M ) and SIM-A9 microglia ( N ) expressing shC or sh Gpnmb for 24 hours. n = 3 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Expressing, Recombinant, shRNA, Control

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) Strategy to cluster GBM samples into GPNMB -low and GPNMB -high subgroups based on 4 published single-cell RNA-Seq (scRNA-Seq) datasets ( GSE131928 , GSE148842 , GSE89567 , and EGAS00001004422). ( B ) GSC signature score in GPNMB -low ( n = 19) and GPNMB -high ( n = 20) subgroups based on the above 4 scRNA-Seq datasets. Student’s t test. ( C and D ) Immunoblots for CD133 and SOX2 in lysates of GSC272 ( C ) and GSC2 ( D ) cells treated with GPNMB recombinant protein at the different indicated concentrations for 24 hours. ( E and F ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells treated with the conditioned media (CM) of THP-1 macrophages ( E ) and HMC3 microglia ( F ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ) for 24 hours. ( G and H ) Immunoblots for CD133 and SOX2 in lysates of GSC2 cells treated with the CM of THP-1 macrophages ( G ) and HMC3 microglia ( H ) expressing shC and sh GPNMB for 24 hours. ( I – L ) Representative images and quantification of tumorspheres of GSC272 cells treated with the CM of THP-1 macrophages ( I and J ) or HMC3 microglia ( K and L ) expressing shC and sh GPNMB for 2 weeks. Scale bar: 200 μm. n = 5 independent samples. One-way ANOVA test. ( M – P ) Representative images and quantification of tumorspheres of GSC2 cells treated with the CM of THP-1 macrophages ( M and N ) or HMC3 microglia ( O and P ) expressing shC and sh GPNMB for 2 weeks. Scale bar: 200 μm. n = 5 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Western Blot, Recombinant, Expressing, shRNA, Control

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A and B ) Representative images ( A ) and quantification ( B ) of human phospho-kinases in GSC2 cells treated with or without GPNMB recombinant protein (100 ng/mL) for 1 hour. Affected kinases are indicated. ( C and D ) Immunoblots for P-RSK2, RSK2, P-PYK2, and PYK2 in lysates of GSC272 cells treated with GPNMB recombinant protein at the indicated concentrations and time points. ( E ) Extracellular acidification rate (ECAR) of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PYK2 inhibitor PF-271 (15 nM) or RSK1/2 inhibitor SL0101 (100 μM) for 24 hours. n = 6 independent samples. ( F ) Relative L-lactate levels in GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 3 independent samples. One-way ANOVA test. ( G ) ECAR of CT2A cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 6 independent samples. ( H ) Relative L-lactate levels in CT2A cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM) or SL0101 (100 μM) for 24 hours. n = 3 independent samples. One-way ANOVA test. ( I and J ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM, I ) or SL0101 (100 μM, J ) for 24 hours. ( K – N ) Tumorsphere formation assays of GSC272 cells treated with GPNMB recombinant protein (100 ng/mL) in the presence or absence of PF-271 (15 nM, K and L) or SL0101 (100 μM, M and N ) for 2 weeks. Scale bar: 200 μm. n = 3–5 independent samples. One-way ANOVA test. ** P < 0.01, *** P < 0.001, n.s., not significant ( P > 0.05).

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Recombinant, Western Blot

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) T-distributed stochastic neighbor embedding (t-SNE) dimensional reduction of cancer cells and immune cells from GBM tumor samples based on single-cell RNA-Seq (scRNA-Seq) dataset ( GSE182109 ). ( B ) Pattern representing single-cell gene expression (left) and quantification (right) of GPNMB in TAMs and CD44 in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). ( C ) Relationship between GPNMB expression in TAMs and CD44 expression in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). P and r values are shown. Pearson’s test. ( D ) Relationship between GPNMB expression in IMCs and CD44 expression in GSCs/GBM cells based on scRNA-Seq dataset ( GSE182109 ). P and r values are shown. Pearson’s test. ( E ) Immunoblots for CD44 in lysates of GSC272 cells expressing shRNA control (shC) or CD44 shRNA (sh CD44 ). ( F ) Immunoblots for P-RSK2, RSK2, P-PYK2, and PYK2 in lysates of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 2 hours. ( G ) Extracellular acidification rate (ECAR) of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 24 hours. ECAR was obtained from the Seahorse experiments, and glucose was added at the indicated time point. n = 3 independent samples. ( H ) Immunoblots for CD133 and SOX2 in lysates of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 24 hours. ( I and J ) Representative images ( I ) and quantification ( J ) of tumorspheres of GSC272 cells expressing shC and sh CD44 treated with or without GPNMB recombinant protein (100 ng/mL) for 2 weeks. Scale bar: 200 μm. n = 4 independent samples. One-way ANOVA test. ** P < 0.01, *** P < 0.001, n.s., not significant ( P > 0.05).

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: RNA Sequencing, Gene Expression, Expressing, Western Blot, shRNA, Control, Recombinant

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A and B ) Survival curves of nude mice coimplanted with GSC272 cells (1 × 10 5 cells/mouse) and THP-1 macrophages (1 × 10 5 cells/mouse, A ) or HMC3 microglia (1 × 10 5 cells/mouse, B ) expressing shRNA control (shC) or GPNMB shRNA (sh GPNMB ). n = 9–10 mice per group. Median survival days are shown. Log-rank test. ( C and D ) Survival curves of C57BL/6 mice coimplanted with CT2A cells (1 × 10 4 cells/mouse) and Raw264.7 macrophages (1 × 10 4 cells/mouse, C ) or SIM-A9 microglia (1 × 10 4 cells/mouse, D ) expressing shC or sh Gpnmb . n = 7 mice per group. Median survival days are shown. Log-rank test. ( E – H ) Representative images and quantification of immunofluorescence staining of SOX2 in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and THP-1 macrophages ( E and F ) or HMC3 microglia ( G and H ) expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. ( I – K ) Representative images ( I ) and quantification ( J and K ) of immunofluorescence staining of Ki67 ( I and J ) and CC3 ( I and K ) in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and THP-1 macrophages expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. ( L – N ) Representative images ( L ) and quantification ( M and N ) of immunofluorescence staining of Ki67 ( L and M ) and CC3 ( L and N ) in tumors from the brains of nude mice intracranially coimplanted with GSC272 cells and HMC3 microglia expressing shC or sh GPNMB . Scale bar: 50 μm. n = 3 independent samples. One-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Expressing, shRNA, Control, Immunofluorescence, Staining

Journal: JCI Insight

Article Title: Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

doi: 10.1172/jci.insight.187684

Figure Lengend Snippet: ( A ) GPNMB expression levels in different types of immune cells from GBM tumors based on single-cell RNA-Seq (scRNA-Seq) dataset (EGAS00001004871). ( B ) Pattern representing single-cell gene expression of GPNMB in macrophages, microglia, M-IMCs, and PNM-IMCs from GBM tumors based on scRNA-Seq dataset (EGAS00001004871). ( C ) Coimmunofluorescence staining for GPNMB (red) and F4/80 (macrophage marker, green) or CX3CR1 (microglia marker, green) in human GBM tumor samples. Scale bar: 50 μm. ( D ) Correlation between GPNMB expression and macrophage/microglia signature scores in IDH1-WT TCGA samples from patients with GBM. P and r values are shown. Pearson’s test. ( E ) The expression of GPNMB in macrophage-high ( n = 185) and macrophage-low ( n = 187) as well as microglia-high ( n = 185) and microglia-low ( n = 187) groups of IDH1-WT TCGA samples from patients with GBM. Student’s t test. ( F and G ) Representative images ( F ) and correlation quantification analysis ( G ) between GPNMB and SOX2 expression in human GBM tumor samples ( n = 30) based on immunofluorescence staining. Scale bar: 50 μm. P and r values are shown. Pearson’s test. ( H ) Relationship between histological Ki67 score in tumors and GPNMB expression level in the plasma of patients with GBM ( n = 36). P and r values are shown. Pearson’s test. (I and J) Survival curves of patients with GBM with high and low GPNMB expression in IDH1-WT TCGA (I) and Gravendeel (J) GBM datasets from GlioVis ( https://gliovis.bioinfo.cnio.es/ ). Log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After washing, cells were continuously cultured for 3–5 days with GPNMB recombinant protein treatment (R&D Systems, 2550-AC) or macrophage/microglia-derived CM in the dark and used for flow cytometry analysis.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining, Marker, Immunofluorescence, Clinical Proteomics