p ret  (Cell Signaling Technology Inc)

Journal: International Journal of Oncology

Article Title: GDNF regulates lipid metabolism and glioma growth through RET/ERK/HIF-1/SREBP-1

doi: 10.3892/ijo.2022.5399

Figure Lengend Snippet: GDNF/RET signaling is upregulated in glioma and promote lipid metabolism. mRNA expression of GDNF normal brain tissues and glioma (A) RT-qPCR and (C) TCGA data analysis. (B) Association between GDNF expression and degree of malignancy (RT-qPCR). (D) Survival curves for patients with low and high GDNF mRNA expression levels in glioma (TCGA database). Western blot analysis of total cell lysates (FASN, SCD1, ACC) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM complete medium with (E) different dose of GDNF for 48 h or (F) with 50 ng/ml GDNF at indicated times. (G) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells were cultured in DMEM complete medium and treated with 50 ng/ml GDNF for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity with ImageJ (n=20). Images were captured at x200 magnification. (H) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM complete medium with or without GDNF (50 ng/ml) for 24 h. (I) SREBP-1 mRNA expression in normal brain tissues and glioma (TCGA data analysis). (J) Survival curves for patients with low and high SREBP-1 mRNA expression levels in glioma (TCGA database). (K) Western blot analysis of p-RET, p-ERK and nSREBP-1 levels in U251 and U87 glioma cells that were cultured in DMEM complete medium with or without 50 ng/ml GDNF in the presence or absence of 20 μ M RPI-1 (GDNF/RET inhibitor) for 48 h. (L) U251 and U87 glioma cells cultured in DMEM complete medium with different dose of GDNF for 48 h or with 50 ng/ml GDNF at indicated times. Relative viability of glioma cells detected by MTT assay. (M) Relative viability of glioma cell cultured in DMEM complete medium with 50 ng/ml GDNF in the presence or absence of 20 μ M RPI-1 for 48 h. Significance was determined by unpaired Student's t test ( * P<0.01, *** P<0.001, **** P<0.0001). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; FASN, fatty acid synthase; SCD1, stearoylCoA desaturase-1; ACC, acetyl CoA carboxylase; SREBP-1, sterol regulatory element-binding protein-1; p-, phosphorylated.

Article Snippet: After blocking with 5% skim milk at room temperature for 2 h, the membranes were probed with primary antibodies against acetyl CoA carboxylase (ACC; 1:1,000; cat. no. 3662, Cell Signaling Technology, Inc.), HIF-1 (1:1,000; cat. no. 36169, Cell Signaling Technology, Inc.), SCAP (1:1,000; cat. no. 13102, Cell Signaling Technology, Inc.), SREBP-1 (1:1,000; cat. no. sc-365513, Santa Cruz Biotechnology, Inc.), fatty acid synthase (FASN) (1:1,000; cat. no. 3180, Cell Signaling Technology, Inc.), RET (1:1,000; cat. no. 14556, Cell Signaling Technology, Inc.), phosphorylated (p)-RET (1:1,000; cat. no. SAB4504530, MilliporeSigma), ERK (1:1,000; cat. no. 5013, Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 4370, Cell Signaling Technology, Inc.), stearoylCoA desaturase-1 (SCD1) (1:1,000; cat. no. 2794, Cell Signaling Technology, Inc.), β-tubulin (1:1,000; cat. no. 2128, Cell Signaling Technology, Inc.) and lamin B (1:1,000; cat. no. 13435, Cell Signaling Technology, Inc.), at 4°C for 12 h. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. ZB-2305, ZSGB-BIO; OriGene Technologies, Inc.) secondary antibodies at room temperature for 2 h. Immunoreactivity was visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Immunofluorescence, Staining, MTT Assay, Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Binding Assay

Journal: International Journal of Oncology

Article Title: GDNF regulates lipid metabolism and glioma growth through RET/ERK/HIF-1/SREBP-1

doi: 10.3892/ijo.2022.5399

Figure Lengend Snippet: GDNF/RET signaling pathway promotes glucose absorption and subsequently activates SREBP-1 through HBP. U251 and U87 glioma cells cultured in DMEM complete medium with (A) different dose of GDNF for 48 h or with (B) 50 ng/ml GDNF at indicated times and (C) in the presence or absence of 20 μ M RPI-1 (GDNF/RET inhibitor). Glucose uptake ability of glioma cells evaluated by fluorescent glucose 2-NBDG. (D and E) Western blot analysis of total cell lysates (RET/p-RET, ERK/p-RET) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with 50 ng/ml GDNF, 10 mM glucose or in combination with 20 μ M RPI-1 for 48 h. (F) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells cultured in DMEM glucose-free medium and treated with 50 ng/ml GDNF or 25 mM glucose or in combination with 20 μ M RPI-1 for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity using ImageJ (n=20). Images were captured at x200 magnification. (G) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM glucose-free medium and treated with 50 ng/ml GDNF, 10 mM glucose, or 20 μ M RPI-1 for 24 h. (H and I) Western blot analysis of nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium with or without 10 mM glucose, 10 mM lactate, 10 mM pyruvate, or 20 mM HBP or in combination with 20 μ M azaserine (HBP inhibitor) for 48 h. (J) Western blot analysis of total cell lysates (RET/p-RET) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with or without GDNF or in combination with 20 μ M RPI-1 or 20 mM HBP for 48 h. (K) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM complete medium and treated with 50 ng/ml GDNF and in the presence or absence of 20 μ M azaserine for 24 h. (L) U251 and U87 glioma cells cultured in DMEM complete medium treated with 50 ng/ml GDNF and in combination with 20 μ M azaserine or 20 mM HBP for 48 h. Relative viability of glioma cells detected by MTT assay. ( * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001; NS : not significant). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; SREBP-1, sterol regulatory element-binding protein-1; HBP, N-acetylglucosamine; p-, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: After blocking with 5% skim milk at room temperature for 2 h, the membranes were probed with primary antibodies against acetyl CoA carboxylase (ACC; 1:1,000; cat. no. 3662, Cell Signaling Technology, Inc.), HIF-1 (1:1,000; cat. no. 36169, Cell Signaling Technology, Inc.), SCAP (1:1,000; cat. no. 13102, Cell Signaling Technology, Inc.), SREBP-1 (1:1,000; cat. no. sc-365513, Santa Cruz Biotechnology, Inc.), fatty acid synthase (FASN) (1:1,000; cat. no. 3180, Cell Signaling Technology, Inc.), RET (1:1,000; cat. no. 14556, Cell Signaling Technology, Inc.), phosphorylated (p)-RET (1:1,000; cat. no. SAB4504530, MilliporeSigma), ERK (1:1,000; cat. no. 5013, Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 4370, Cell Signaling Technology, Inc.), stearoylCoA desaturase-1 (SCD1) (1:1,000; cat. no. 2794, Cell Signaling Technology, Inc.), β-tubulin (1:1,000; cat. no. 2128, Cell Signaling Technology, Inc.) and lamin B (1:1,000; cat. no. 13435, Cell Signaling Technology, Inc.), at 4°C for 12 h. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. ZB-2305, ZSGB-BIO; OriGene Technologies, Inc.) secondary antibodies at room temperature for 2 h. Immunoreactivity was visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Cell Culture, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, MTT Assay, Derivative Assay, Transfection, Binding Assay, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: GDNF regulates lipid metabolism and glioma growth through RET/ERK/HIF-1/SREBP-1

doi: 10.3892/ijo.2022.5399

Figure Lengend Snippet: GDNF/RET Signaling promotes glucose absorption by upregulating HIF-1. (A) Western blot analysis of p-RET, HIF-1 levels in U251 and U87 glioma cells that were cultured in DMEM complete medium with or without 50 ng/ml GDNF in the presence or absence of 20 μ M RPI-1 (GDNF/RET inhibitor) for 48 h. U251 and U87 glioma cells were transfected with siHIF-1 for 48 h and cultured in DMEM complete medium with or without 50 ng/ml GDNF; (B) Glucose uptake ability of glioma cells was evaluated by fluorescent glucose 2-NBDG; (C) Protein expression determined by western blotting; (D) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) and quantitative evaluation of SREBP-1 nuclear intensity with ImageJ (n=20). Images were captured at x200 magnification. (E) Reverse transcription-quantitative PCR analysis of mRNA levels in U87 and U251 glioma cells following knock down of HIF-1 and cultured in DMEM complete medium with or without 50 ng/ml GDNF for 24 h. U251 and U87 glioma cells transfected with siHIF for 48 h and cultured in DMEM complete medium with 50 ng/ml GDNF or 20 mM HBP. (F) Protein expression was determined by western blotting. (G) Relative viability of glioma cells detected by MTT assay. Significance was determined by unpaired Student's t test ( * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001; NS : not significant). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; HIF-1, hypoxia-inducible factor 1; p-, phosphorylated; RET, rearranged during transfection; HBP, N-acetylglucosamine.

Article Snippet: After blocking with 5% skim milk at room temperature for 2 h, the membranes were probed with primary antibodies against acetyl CoA carboxylase (ACC; 1:1,000; cat. no. 3662, Cell Signaling Technology, Inc.), HIF-1 (1:1,000; cat. no. 36169, Cell Signaling Technology, Inc.), SCAP (1:1,000; cat. no. 13102, Cell Signaling Technology, Inc.), SREBP-1 (1:1,000; cat. no. sc-365513, Santa Cruz Biotechnology, Inc.), fatty acid synthase (FASN) (1:1,000; cat. no. 3180, Cell Signaling Technology, Inc.), RET (1:1,000; cat. no. 14556, Cell Signaling Technology, Inc.), phosphorylated (p)-RET (1:1,000; cat. no. SAB4504530, MilliporeSigma), ERK (1:1,000; cat. no. 5013, Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 4370, Cell Signaling Technology, Inc.), stearoylCoA desaturase-1 (SCD1) (1:1,000; cat. no. 2794, Cell Signaling Technology, Inc.), β-tubulin (1:1,000; cat. no. 2128, Cell Signaling Technology, Inc.) and lamin B (1:1,000; cat. no. 13435, Cell Signaling Technology, Inc.), at 4°C for 12 h. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. ZB-2305, ZSGB-BIO; OriGene Technologies, Inc.) secondary antibodies at room temperature for 2 h. Immunoreactivity was visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Western Blot, Cell Culture, Transfection, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, MTT Assay, Derivative Assay