Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 3 | P7C3 reduces the protein level of PGK1 and PGK2. (A) Representative spots of P7C3 target proteins, including the glycolytic process. (B) Western blotting for detecting protein levels of P7C3 target proteins that are related to glycolysis after U87MG and U118MG cells were treated with P7C3 at a concentration of 0 mM, 30 mM, and 50 mM for 24 h. (C) PGK1 activity levels in P7C3-treated U87MG and U118MG cells at a concentration of 0 mM, 30 mM, and 50 mM (n = 4). (D) The expression profiles of PGK1 and PGK2 mRNA levels. Data were obtained from the Human Protein Atlas database. The images are available at: https://www. proteinatlas.org/ENSG00000102144-PGK1/cell, and (https://www.proteinatlas.rg/ENSG00000170950-PGK2/cell). (E) Statistical analysis of PGK1 mRNA level among WHO grade II-IV glioma. The data were based on TCGA and CGGA and are available at: http://gliovis.bioinfo.cnio.es/. (F) Kaplan–Meier survival analysis of PGK1 mRNA level in glioma patients. The data were based on TCGA and CGGA and are available at: http://gliovis.bioinfo.cnio.es/. (The data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Western Blot, Concentration Assay, Activity Assay, Expressing

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 4 | P7C3 directly binds to PGK1. (A) Streptavidin affinity assay for P7C3-Bio binds to PGK1 in P7C3-treated glioma cells. Cells in culture were treated with 50 mM P7C3-Bio for 24 h, and then streptavidin agarose was added to incubate with the cell lysates. (B) Streptavidin affinity assay for P7C3-Bio binds to PGK1 upon cell lysates. Cell lysates were incubated with 10 mM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (C) Streptavidin affinity assay for P7C3-Bio binds to purified PGK1 protein. In total, 25 mg recombinant human PGK1 solution was incubated with 10 mM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (D) Motif analysis based on top 100 P7C3 target proteins was performed by MEME, and three motifs were identified. The motif consensus and motif locations at PGK1 are shown. (E) The P7C3 binding sites of PGK1 were identified by ESI-LC-MSMS. The P7C3 binding sites were identified by the predicted peptide mass plus P7C3 [C21H18Br2N2O with a loss of a water] and minus one H.

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Incubation, Recombinant, Binding Assay

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 5 | P7C3 accelerates PGK1 protein degradation. (A) PGK1 mRNA level was measured by RT-PCR. P7C3 upregulates the PGK1 mRNA levels in U87MG and U118MG cells (n = 3). (B) Time-course assay of P7C3 effects on PGK1 degradation. U87MG and U118MG cells were treated by CHX (100 ug/mL) with or without P7C3 (50 mM) for 24 h, and then PGK1 protein levels were detected by western blotting. (C) Protein level curve for PGK1 based on time-course analysis of U87MG and U118MG cells (n = 3). (D) Western blotting of PGK1 protein degradation pathway. MG132 (5 mM) and CQ (20 mM) were used to treat cells for 6 h, followed by P7C3 treatment for another 18 h, and the protein level of PGK1 was detected by western blotting. (E) The statistical analysis of the quantized protein levels of PGK1 in U87MG and U118MG cells (n = 3). (F) Transmission electron microscopy assay after glioma cells were treated with P7C3 for 24 hours. Autophagosomes, autolysosomes, and phagophores are indicated by yellow, green, and red arrows (scale bar: original mage, 5 mm; Zoom image, 1 mm). (G) Western blotting for two key autophagy-associated proteins Beclin-1, and LC3A/B, after U87MG and U118MG cells were treated with 0 mM, 30 mM, and 50 mM P7C3 for 24 h. (Data are expressed as mean ± SD, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means none significance).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 7 | P7C3 inhibits glioma in vivo. (A) Representative in vivo IVIS Luc fluorescence images of xenografts mice on days 7 and 21 after injection of U87MG cells. (B) Statistical result of photon flux in xenografts mice on days 7 and 21 (n = 12). (C) Kaplan–Meier curves of survival rate of xenografts mice (n = 6). (D) Immunohistochemical staining of PGK1 in the indicated tumor tissues was performed, PGK1 positive cells were stained brown (scale bar: 20 mm). (E) Immunohistochemical staining of Ki67 in the indicated tumor tissues was performed. Ki67 positive cells were stained brown (scale bar: 50 mm). (F) TUNEL analyses of the indicated tumor tissues were performed. Apoptotic cells were stained brown (scale bar: 50 mm). (G) Immunohistochemical staining of labeling astrocytes, microglia, and oligodendrocytes in the mice brain by using GFAP, Iba1, and SOX10, respectively (scale bar: 100 mm). (H) Statistical analysis of GFAP-positive, Iba1- positive, and SOX10-positive cells in the mouse brain (n = 3). (The data were expressed as mean ± SD, nsP > 0.05, *P < 0.05, ns means none significance).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: In Vivo, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Labeling

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: NEAT1 directly interacts with PGK1. A Coomassie brilliant blue staining of NEAT1 pulldown. Asterisks show different bands between the sense and antisense lanes. B List of the top 10 differentially expressed proteins identified by mass spectrometry. C Confocal images showing subcellular localization of NEAT1 and PGK1 in U251 and T98G cells. D NEAT1 pull-down followed by western blot exhibited the binding of NEAT1 to PGK1. E RIP assay showed the binding of NEAT1 to PGK1. F CCK-8 analysis of U251 and T98G cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. G Colony formation assays of U251 and T98G cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. H Western blot analysis of CDK2, CDK4, and CDK6 in U251 and T98G cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. I , J ECAR was measured through the Glycolysis Stress in U251 cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. K , L ECAR was measured through the Glycolysis Stress in T98G cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. M Lactate concentration was assessed in U251 and T98G cells transfected with sh-ctrl or sh-NEAT1. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD of triple independent experiments

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: Staining, Mass Spectrometry, Western Blot, Binding Assay, CCK-8 Assay, Transfection, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: NEAT1 inhibits PGK1 degradation via the ubiquitin–proteasome pathway. A Relative PGK1 mRNA levels in U251 cells transfected with sh-ctrl or sh-NEAT1. B Relative PGK1 mRNA levels in T98G cells transfected with sh-ctrl or sh-NEAT1. C Western blot analysis of PGK1 in U251 and T98G cells transfected with sh-NEAT1 or NEAT1. D Western blot analysis of PGK1 in transfected U251 and T98G cells treated with MG132 for the indicated time. E Western blot analysis of PGK1 in transfected U251 cells treated with Chx for the indicated time (left). The quantification of PGK1 degradation rate by gray scale analysis (right). F Western blot analysis of PGK1 in transfected T98G cells treated with Chx for the indicated time (left). The quantification of PGK1 degradation rate by gray scale analysis (right). G Ubiquitinated PGK1 detected by immunoprecipitation with anti-Flag antibody in transfected U251 cells. H Ubiquitinated PGK1 detected by immunoprecipitation with anti-Flag antibody in transfected T98G cells. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD of triple independent experiments

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Immunoprecipitation

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: Hairpin A of NEAT1 interacts with PGK1. A A schema of truncated NEAT1. B RNA pull-down via sequential truncated NEAT1 fragments showed the binding region of NEAT1 with PGK1. C The structure of NEAT1 predicted by RNAfold indicates a stable stem-loop (hairpin A) structure within 1–500 nt. D The deletion of hairpin A abolished the binding of NEAT1 with PGK1. E RIP assays performed after the deletion of hairpin A in U251 and T98G cells. F CCK-8 analysis of U251 and T98G cells transfected with WT or hairpin A-deleted NEAT1. G Colony formation assays of U251 and T98G cells transfected with WT or hairpin A-deleted NEAT1. H Western blot analysis of CDK2, CDK4, and CDK6 in U251 and T98G cells transfected with WT or hairpin A-deleted NEAT1. J ECAR was measured through the Glycolysis Stress in U251 cells transfected with WT or hairpin A-deleted NEAT1. I ECAR was measured through the Glycolysis Stress in T98G cells transfected with WT or hairpin A-deleted NEAT1. K Lactate concentration was assessed in U251 and T98G cells transfected with WT or hairpin A-deleted NEAT1. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD of triple independent experiments

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: Binding Assay, CCK-8 Assay, Transfection, Western Blot, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: NEAT1 interacts with M1 residues of PGK1. A A schema of truncated PGK1. B Western blot analysis of U251 and T98G cells transfected with full length or truncated Flag-PGK1. C RIP assay showed the binding of NEAT1 to M1 residues of PGK1. D The deletion of M1 abolished the binding of PGK1 with NEAT1. E CCK-8 analysis of U251 and T98G cells transfected with full length or M1-deleted PGK1. F Colony formation assays of U251 and T98G cells transfected with full length or M1-deleted PGK1. G Western blot analysis of CDK2, CDK4, and CDK6 in U251 and T98G cells transfected with full length or M1-deleted PGK1. H ECAR was measured through the Glycolysis Stress in U251 cells transfected with full length or M1-deleted PGK1. I ECAR was measured through the Glycolysis Stress in T98G cells transfected with full length or M1-deleted PGK1. J Lactate concentration was assessed in U251 and T98G cells transfected with full length or M1-deleted PGK1. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD of triple independent experiments

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: Western Blot, Transfection, Binding Assay, CCK-8 Assay, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: NEAT1/PGK1 promotes tumor progression in vivo. A Pseudocolor bioluminescence images of orthotopic tumors derived from U251 cells transfected with sh-NEAT1 or co-transfected with sh-NEAT1 and PGK1. B Bioluminescence was quantified in tumors from three groups. C Survival curves of mice from three groups. D Immunohistochemical analysis of ki-67 and PGK1 in tumors from three groups. E Three dimensional scatter plot of NEAT1, ki-67, and PGK1 in tumors from three groups. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: In Vivo, Derivative Assay, Transfection, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet: Characterization of NEAT1 as a biomarker for progression of glioma. A The CGGA database was used to analyze the expression of NEAT1 in different grade glioma samples. B The TCGA database was used to analyze the expression of NEAT1 in tumor adjacent tissues and GBM samples. C Twenty-five clinical samples were used to analyze the expression of NEAT1 in tumor adjacent tissues and GBM samples. D Overall survival (OS) of GBM patients with high or low NEAT1 expression from CGGA database. E Overall survival (OS) of GBM patients with high or low NEAT1 expression from TCGA database. F Overall survival (OS) of GBM patients with high or low NEAT1 expression from clinical samples. G The correlation between NEAT1 and PGK1 in GBM samples from CGGA database. H The correlation between NEAT1 and PGK1 in GBM samples from TCGA database. I PGK1 expression levels in GBM clinical samples with high or low NEAT1 levels. * p < 0.05, ** p < 0.01. Error bars indicate mean ± SD

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: Biomarker Discovery, Expressing

Journal: Journal of Translational Medicine

Article Title: LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1

doi: 10.1186/s12967-022-03273-2

Figure Lengend Snippet:

Article Snippet: Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study.

Techniques: