Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Comparison of CHSY1 gene expression in glioma subtypes and normal brain tissue in the REMBRANDT glioma microarray database. ** P < 0.01, **** P < 0.0001. b High expression of CHSY1 was associated with worse overall survival in glioma patients. The high and low expression groups were divided by median expression level of CHSY1 in 329 cases. These data were from the REMBRANDT database ( http://www.betastasis.com/glioma/rembrandt/ ). c Immunohistochemistry of CHSY1 (upper panel) and CS56 (lower panel) on tissue array contains 85 primary glioma cases. The staining was visualized in brown color with a 3,3-diaminobenzidine liquid substrate system. All sections were counterstained with hematoxylin. Representative images of four glioma cases with different staining intensities are shown. Amplified images are shown at the bottom right of each image. Scale bars, 50 μm. Arrows indicate positive stained glioma cells. d Representative images of CHSY1 staining on normal brain tissue ( n = 5). e Statistical analysis of immunohistochemistry in glioma tissue array. Mann–Whitney U -test was used. P -values are shown at top. f Expression of CHSY1 in glioma cell lines and normal human brain tissue. The protein expression was analyzed by western blotting. Total loading protein is shown at bottom. Relative expression levels to total brain tissue form three independent blots are shown at the right.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Amplification, MANN-WHITNEY, Western Blot

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Stable overexpression of CHSY1 in GL261 cell and siRNA knockdown of CHSY1 in A172 and U118 cells. The CHSY1 expression were analyzed by western blots. ACTB was used as loading control. Control-siRNA (Ctr si) CHSY1-siRNA (CHSY1 si). b Surface CS56 antibody staining on GL261, A172, and U118 transfectants were analyzed by flow cytometry with anti-mouse IgM-FITC. Nonspecific mouse IgM was used as an iso-type control (iso). Representative images are shown. Results of CS56 flow cytometry are presented as the geometric mean fluorescence intensity (Geo-MFI) ± SD from three experiments. ** P < 0.01. c Immunofluorescence microscopy analysis of CHSY1 expression (red) and CS56 immunoreactivity (green) in control and CHSY1 siRNA-transfected A172 cells. Amplified images are shown at the right. Nuclei were counterstained with DAPI (blue), scale bar, 25 μm. d CHSY1 modulated cell viability in vitro. Cell viability of GL261, A172, and U118 cells was measured using CCK8 assays at indicted time points. Data were represented as means ± SD from three independent experiments. ** P < 0.01. e Overexpression of CHSY1-enhanced tumor growth in vivo. GL261 transfectants were orthotopically injected into right cerebral hemisphere C57BL/6 mice ( n = 5 for each group). Mouse brain was excised and the size of tumors was measured and shown as means ± SD. * P < 0.05. Gross brain images of superior view and inferior view are shown (middle). Blue dash lines indicate location of tumors. Represented images of H-E stain of brain section are shown. Scale bar, 2.0 mm. f CHSY1 promotes cell proliferation in GL261 tumor. Cell proliferation of tumor cells was evaluated by immunofluorescence staining for Ki67 and representative images of tumor tissue of brain section are shown (bottom). Results are presented as the mean ± SD from three fields of each section. ** P < 0.01. Scale bar 100 μm.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Over Expression, Expressing, Western Blot, Staining, Flow Cytometry, Fluorescence, Immunofluorescence, Microscopy, Transfection, Amplification, In Vitro, In Vivo, Injection

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a p-RTK array showing the effects of CHSY1 on the phosphorylation of RTKs. Cells were starved for 3 h and then stimulated with FBS (10%) for 15 min. Cell lysates of mock and CHSY1-overexpressed GL261 cells (left); control and CHSY1-silenced A172 cells (right) were applied to p-RTK arrays including 39 RTKs. b CHSY1 modulates PDGF-induced signaling in glioblastoma cells. GL261 and A172 transfectants were starved for 3 h and then treated with (+)/without (−) PDGF-AB (20 ng/mL) for 5 and 15 min. Cell lysates (20 μg) were analyzed by western blotting with various antibodies, as indicated. c CHSY1 regulates PDGFRA protein levels in glioblastoma cells. Relative PDGFRA levels of western blottings with or without PDGF-AB stimulation are quantified and shown as mean ± SD from three experiments. d Immunofluorescence microscopy analysis of PDGFRA expression (green) in GL261 brain tumor sections. Amplified images are shown at the right. Nuclei were counterstained with DAPI (blue), scale bar, 60 μm. e Knockdown of CHSY1 suppresses PDGF-induced cell viability. Control and CHSY1-silenced A172 cells were cultured in low-serum condition (1% of FBS) and treated with PDGF (20 ng/ml) or EGF (20 ng/ml). Cell viability was measured by CCK8 assays at. Data were represented as means ± SD from three independent experiments. ** P < 0.01; ns: nonsignificant. f Knockdown of CHSY1 suppresses PDGF-induced cell proliferation. Cells were cultured in low-serum condition (1% of FBS) and treated with PDGF (20 ng/ml) or EGF (20 ng/ml) for 48 h. Cells were immunofluorescently stained for Ki67 and Ki67-positive cells were counted under a microscope. Results are presented as means ± SD from three independent experiments. ** P < 0.01, ns: nonsignificant. g Western blot analysis of PDGFRA protein levels in GL261 and A172 transfectants. Cells were treated with 20 μM cycloheximide (CHX) in culture medium at indicated intervals and analyzed by western blotting. Intensities of PDGFRA protein were quantified using a densitometer. h Effects of CHSY1 on surface expression of PDGFRA in GL261 cells and A172 cells. Surface PDGFRA staining after treating cycloheximide at indicated intervals were analyzed by flow cytometry. Nonspecific rabbit IgG was used as an iso-type control (iso). Representative images are shown (left). Statistic results are shown as Geo-MFI ± SD from three experiments (middle). To quantify PDGFRA turnover rate, Geo-MFI of iso-type control was subtracted and the relative PDGFRA intensities to the 0 h of each group are shown at the right. * P < 0.05, ** P < 0.01.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Western Blot, Immunofluorescence, Microscopy, Expressing, Amplification, Cell Culture, Staining, Flow Cytometry

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Effects of crenolanib, a PDGFRA inhibitor, on CHSY1-enhanced cell viability. GL261 transfectants were treated with solvent (0.005% of ethanol) or crenolanib (0.5 μM) and then analyzed by CCK8 assays at 72 h. Data are represented as means ± SD from three independent experiments. ** P < 0.01, *** P < 0.001. b Effects of PDGFRA inhibitor on anchorage-dependent colony formation. Results were presented as means ± SD from three independent experiments. Representative images were shown at the bottom. *** P < 0.001. c Survival analysis of crenolanib treatment in an orthotopic murine model of GL261 glioma. Mock and CHSY1-overexpressing transfecants were implanted into C57BL/6 mice. The next day post tumor implant, mice were treated with 15 mg/kg of crenolanib (Cre) via daily intraperitoneal injection for 7 days. Equivalent 5% of glycerol formal (Gly) was taken as solvent control. Survival was assessed via Kaplan–Meir survival curves. ** P < 0.01 (CHSY1-Gly vs. CHSY1-Cre ) . # P < 0.05 (Mock-Cre vs. CHSY1-Cre). Median survival time (MS) of each group is shown at the right.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Injection

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Comparison of CHSY1 gene expression in glioma subtypes and normal brain tissue in the REMBRANDT glioma microarray database. ** P < 0.01, **** P < 0.0001. b High expression of CHSY1 was associated with worse overall survival in glioma patients. The high and low expression groups were divided by median expression level of CHSY1 in 329 cases. These data were from the REMBRANDT database ( http://www.betastasis.com/glioma/rembrandt/ ). c Immunohistochemistry of CHSY1 (upper panel) and CS56 (lower panel) on tissue array contains 85 primary glioma cases. The staining was visualized in brown color with a 3,3-diaminobenzidine liquid substrate system. All sections were counterstained with hematoxylin. Representative images of four glioma cases with different staining intensities are shown. Amplified images are shown at the bottom right of each image. Scale bars, 50 μm. Arrows indicate positive stained glioma cells. d Representative images of CHSY1 staining on normal brain tissue ( n = 5). e Statistical analysis of immunohistochemistry in glioma tissue array. Mann–Whitney U -test was used. P -values are shown at top. f Expression of CHSY1 in glioma cell lines and normal human brain tissue. The protein expression was analyzed by western blotting. Total loading protein is shown at bottom. Relative expression levels to total brain tissue form three independent blots are shown at the right.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Amplification, MANN-WHITNEY, Western Blot

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Stable overexpression of CHSY1 in GL261 cell and siRNA knockdown of CHSY1 in A172 and U118 cells. The CHSY1 expression were analyzed by western blots. ACTB was used as loading control. Control-siRNA (Ctr si) CHSY1-siRNA (CHSY1 si). b Surface CS56 antibody staining on GL261, A172, and U118 transfectants were analyzed by flow cytometry with anti-mouse IgM-FITC. Nonspecific mouse IgM was used as an iso-type control (iso). Representative images are shown. Results of CS56 flow cytometry are presented as the geometric mean fluorescence intensity (Geo-MFI) ± SD from three experiments. ** P < 0.01. c Immunofluorescence microscopy analysis of CHSY1 expression (red) and CS56 immunoreactivity (green) in control and CHSY1 siRNA-transfected A172 cells. Amplified images are shown at the right. Nuclei were counterstained with DAPI (blue), scale bar, 25 μm. d CHSY1 modulated cell viability in vitro. Cell viability of GL261, A172, and U118 cells was measured using CCK8 assays at indicted time points. Data were represented as means ± SD from three independent experiments. ** P < 0.01. e Overexpression of CHSY1-enhanced tumor growth in vivo. GL261 transfectants were orthotopically injected into right cerebral hemisphere C57BL/6 mice ( n = 5 for each group). Mouse brain was excised and the size of tumors was measured and shown as means ± SD. * P < 0.05. Gross brain images of superior view and inferior view are shown (middle). Blue dash lines indicate location of tumors. Represented images of H-E stain of brain section are shown. Scale bar, 2.0 mm. f CHSY1 promotes cell proliferation in GL261 tumor. Cell proliferation of tumor cells was evaluated by immunofluorescence staining for Ki67 and representative images of tumor tissue of brain section are shown (bottom). Results are presented as the mean ± SD from three fields of each section. ** P < 0.01. Scale bar 100 μm.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Over Expression, Expressing, Western Blot, Staining, Flow Cytometry, Fluorescence, Immunofluorescence, Microscopy, Transfection, Amplification, In Vitro, In Vivo, Injection

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a p-RTK array showing the effects of CHSY1 on the phosphorylation of RTKs. Cells were starved for 3 h and then stimulated with FBS (10%) for 15 min. Cell lysates of mock and CHSY1-overexpressed GL261 cells (left); control and CHSY1-silenced A172 cells (right) were applied to p-RTK arrays including 39 RTKs. b CHSY1 modulates PDGF-induced signaling in glioblastoma cells. GL261 and A172 transfectants were starved for 3 h and then treated with (+)/without (−) PDGF-AB (20 ng/mL) for 5 and 15 min. Cell lysates (20 μg) were analyzed by western blotting with various antibodies, as indicated. c CHSY1 regulates PDGFRA protein levels in glioblastoma cells. Relative PDGFRA levels of western blottings with or without PDGF-AB stimulation are quantified and shown as mean ± SD from three experiments. d Immunofluorescence microscopy analysis of PDGFRA expression (green) in GL261 brain tumor sections. Amplified images are shown at the right. Nuclei were counterstained with DAPI (blue), scale bar, 60 μm. e Knockdown of CHSY1 suppresses PDGF-induced cell viability. Control and CHSY1-silenced A172 cells were cultured in low-serum condition (1% of FBS) and treated with PDGF (20 ng/ml) or EGF (20 ng/ml). Cell viability was measured by CCK8 assays at. Data were represented as means ± SD from three independent experiments. ** P < 0.01; ns: nonsignificant. f Knockdown of CHSY1 suppresses PDGF-induced cell proliferation. Cells were cultured in low-serum condition (1% of FBS) and treated with PDGF (20 ng/ml) or EGF (20 ng/ml) for 48 h. Cells were immunofluorescently stained for Ki67 and Ki67-positive cells were counted under a microscope. Results are presented as means ± SD from three independent experiments. ** P < 0.01, ns: nonsignificant. g Western blot analysis of PDGFRA protein levels in GL261 and A172 transfectants. Cells were treated with 20 μM cycloheximide (CHX) in culture medium at indicated intervals and analyzed by western blotting. Intensities of PDGFRA protein were quantified using a densitometer. h Effects of CHSY1 on surface expression of PDGFRA in GL261 cells and A172 cells. Surface PDGFRA staining after treating cycloheximide at indicated intervals were analyzed by flow cytometry. Nonspecific rabbit IgG was used as an iso-type control (iso). Representative images are shown (left). Statistic results are shown as Geo-MFI ± SD from three experiments (middle). To quantify PDGFRA turnover rate, Geo-MFI of iso-type control was subtracted and the relative PDGFRA intensities to the 0 h of each group are shown at the right. * P < 0.05, ** P < 0.01.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Western Blot, Immunofluorescence, Microscopy, Expressing, Amplification, Cell Culture, Staining, Flow Cytometry

Journal: Oncogenesis

Article Title: Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

doi: 10.1038/s41389-020-0197-0

Figure Lengend Snippet: a Effects of crenolanib, a PDGFRA inhibitor, on CHSY1-enhanced cell viability. GL261 transfectants were treated with solvent (0.005% of ethanol) or crenolanib (0.5 μM) and then analyzed by CCK8 assays at 72 h. Data are represented as means ± SD from three independent experiments. ** P < 0.01, *** P < 0.001. b Effects of PDGFRA inhibitor on anchorage-dependent colony formation. Results were presented as means ± SD from three independent experiments. Representative images were shown at the bottom. *** P < 0.001. c Survival analysis of crenolanib treatment in an orthotopic murine model of GL261 glioma. Mock and CHSY1-overexpressing transfecants were implanted into C57BL/6 mice. The next day post tumor implant, mice were treated with 15 mg/kg of crenolanib (Cre) via daily intraperitoneal injection for 7 days. Equivalent 5% of glycerol formal (Gly) was taken as solvent control. Survival was assessed via Kaplan–Meir survival curves. ** P < 0.01 (CHSY1-Gly vs. CHSY1-Cre ) . # P < 0.05 (Mock-Cre vs. CHSY1-Cre). Median survival time (MS) of each group is shown at the right.

Article Snippet: Full-length CHSY1 cDNA clone and antibody against CHSY1 were purchased from OriGene.

Techniques: Injection