Journal: Experimental animals

Article Title: PGK1 succinylation modulates epileptic seizures and the blood-brain barrier.

doi: 10.1538/expanim.23-0019

Figure Lengend Snippet: Fig. 2. Decreased succinylation of phosphoglycerate kinase (PGK) 1 protein expression levels but not PGK1 is decreased in a rat model of lithium-pilocarpine-induced acute epilepsy. (a) Representative Immunoprecipitation showed significantly decreased succinylation of PGK1 protein expression levels in the hippocampus of the rat model compared with normals (n=3 per group). (b) PGK1 expression levels were not significantly changed in the hippocampus of the rat model compared with normal rats (n=4 per group). All the succinylation of PGK1 protein expression levels were normalized by calculating the OD ratio of succinylation- PGK1 to PGK1 (Ksucc-PGK1/PGK1). All expression levels of PGK1 were normalized by calculating the OD ratio of PGK1 to β-tubulin (PGK1/β-tubulin) (*P<0.05).

Article Snippet: Protein A/G Magnetic beads (MCE, USA) were incubated with anti-PGK1 antibody and rotated overnight at 4°C in a Four-dimensional rotary mixer.

Techniques: Expressing, Immunoprecipitation

Journal: Experimental animals

Article Title: PGK1 succinylation modulates epileptic seizures and the blood-brain barrier.

doi: 10.1538/expanim.23-0019

Figure Lengend Snippet: Fig. 4. The succinylation of phosphoglycerate kinase (PGK) 1 protein expression levels is regulated at K15 in vivo and in vitro. (A) Representative Immunoprecipitation shows that the succinyl- ation of PGK1 protein expression levels was decreased significantly in the K15R variants and increased significantly in the K15E variants compared with the wild-type group by transfecting plasmid in HEK293 cells (n=3 per group). (B) Representative Western blots show that the expression of PGK1 total protein was insignificant in the groups of the K15R variants and the K15E variants compared with the wild-type group by transfecting plasmid in HEK293 cells (n=4 per group). (C) Representative Immunoprecipitation shows that the succinylation of PGK1 protein expression levels decreased significantly in the K15R variants and increased significantly in the K15E variants compared with the scramble group by transfecting with the lentivirus and a rat model of lithium-pilocarpine-induced acute epilepsy (n=3 per group). (D) Representative Western blots show that the expression of PGK1 total protein was insignificant in the groups of the K15R variants and the K15E variants compared with the scramble group by transfecting with the lentivirus and a rat model of lithium-pilocarpine-induced acute epi- lepsy (n=4 per group). All expression levels of the succinylation of PGK1 protein expression levels were normalized by calculating the OD ratio of the succinylation of PGK1 to PGK1 (Ksucc-PGK1/PGK1). All expression levels of PGK1 were normalized by calculating the OD ratio of PGK1 to β-tubulin (PGK1/β-tubulin)(*P<0.05).

Article Snippet: Protein A/G Magnetic beads (MCE, USA) were incubated with anti-PGK1 antibody and rotated overnight at 4°C in a Four-dimensional rotary mixer.

Techniques: Expressing, In Vivo, In Vitro, Immunoprecipitation, Plasmid Preparation, Western Blot

Journal: Experimental animals

Article Title: PGK1 succinylation modulates epileptic seizures and the blood-brain barrier.

doi: 10.1538/expanim.23-0019

Figure Lengend Snippet: Fig. 6. The succinylation of phosphoglycerate kinase (PGK) 1 at K15 is involved in the expression of angiostatin. (a) Representative Western blots show that the expression of angiostatin total protein was decreased significantly in a rat model of lithium-pilocarpine-induced acute epilepsy compared with the normal group (n=4 per group; *P<0.05). (b) Representative Western blots show that the expression of angiostatin total protein was decreased signifi- cantly in the K15R variants and increased significantly in the K15E variants compared with the scramble group by transfecting with the lentivirus and a rat model of lithium-pilocarpine- induced acute epilepsy (n=3 per group; *P<0.05). All expression levels of angiostatin were normalized by calculating the OD ratio of angiostatin to β-tubulin (PGK1/β-tubulin).

Article Snippet: Protein A/G Magnetic beads (MCE, USA) were incubated with anti-PGK1 antibody and rotated overnight at 4°C in a Four-dimensional rotary mixer.

Techniques: Expressing, Western Blot

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MADR + AAVS1 human cell line generation AAVS1 targeting MADR vector was derived from AAVS1-targeting vector AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene 68375).

Techniques: Clone Assay, Recombinant, Lysis, Sequencing, Plasmid Preparation, Mutagenesis, Software

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:

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: Oligonucleotide sequences for qPCR, siRNA, and mutagenesis

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Mutagenesis, shRNA

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: ChIP-Seq peak annotations

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Binding Assay

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, Representative profile of peaks obtained for the Pgk1 gene by ChIP-Seq. ChIP-seq was performed using sPLA 2 -V KD cells transfected with empty vector, sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V, and sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V-H48Q using an anti-Myc-tag mouse monoclonal antibody. Input of sPLA 2 -V KD cells expressing Myc-tagged sPLA 2 -V was used as a ChIP-seq control. ChIP-seq analysis showed a Myc-tagged sPLA 2 -V-binding peak at the upstream region of Pgk1 gene locus. B, ChIP-qPCR validation of Myc-tagged sPLA 2 -V binding to the Pgk1 gene. Data are shown as a percentage expression of input control. The amplification sites (binding site and unrelated site) for PCR are indicated in panel A (ChIP-seq). Each bar represents the mean±SEM of 2–3 independent experiments. C, Promoter assay for Pgk1 gene transcriptional activity. sPLA 2 -V WT cells, sPLA 2 -V KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q were transfected with Cypridina and Renilla luciferase expression vectors. The promoter activity is expressed as the relative luciferase activity normalized to Renilla activity. Values in each bar were normalized to that of WT (=1). Each bar represents the mean±SEM of 6–9 independent experiments. ＊＊ , P ＜0.01 vs. WT. †† , P ＜0.01 vs KD. Upper panel shows a schematic illustration of the promoter construct used in this Cypridina luciferase reporter assay.

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: ChIP-sequencing, ChIP-qPCR, Promoter Assay, Transfection, Plasmid Preparation, Expressing, Control, Binding Assay, Biomarker Discovery, Amplification, Activity Assay, Luciferase, Construct, Reporter Assay

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, Suppression of Beclin1 phosphorylation at S30 (p-Beclin1/Beclin1) by siRNA (#1 and #2)-mediated reduction of PGK1 expression. Values were normalized to that of control siRNA after incubation with PBS as a vehicle (=1). n =5 in each experiment. ＊＊ , P ＜0.01 vs. control siRNA. B, Representative immunoblots for panel A. C, Successful suppression of PGK1 expression by siRNA confirmed by immunoblotting. D, E, F, Comparison of PGK1 expression (D), Beclin1 phosphorylation at S30 (E), and PI3-kinase activity (F) after incubation for 2 hr with 20 µg/mL AcLDL or PBS as a vehicle in sPLA 2 -V-WT and KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q. G, Representative immunoblots showing PGK1 expression and Beclin1 phosphorylation in various types of RAW264.7 cells. Values in panels D and E were normalized to that of WT after incubation with PBS as a vehicle (=1). Each bar represents the mean±SEM of 5–6 independent experiments. ＊ , P ＜0.05, ＊＊ , P ＜0.01 vs. WT, † , P ＜0.05, †† , P ＜0.01 vs. KD.

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Control, Incubation, Western Blot, Comparison

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A and B, siRNA-mediated reduction in PGK1 or Beclin1 expression (#1 and #2) inhibited actin polymerization (A) and translocation of internalized AcLDL conjugated with pHrodo (B). Actin polymerization was assessed by detection of F-actin polymerization with Alexa Fluor 546-phalloidin. Translocation of AcLDL to lysosomes was assessed using flow cytometry of pHrodo-conjugated AcLDL. Details of the methods are described in the text. Each bar represents the mean±SEM of 5 independent experiments. ＊＊ , P ＜0.01 vs. control siRNA. C, Reduction of Beclin1 protein expression by siRNAs (#1 and #2). Values were normalized to that of control siRNA (=1). Upper panel shows representative immunoblots. D and E, Transfection of an expression vector to overexpress PGK1 reversed a decrease in actin polymerization (D) and translocation of internalized AcLDL conjugated with pHrodo (E). Details of methods are described in the text. Each bar represents the mean±SEM of 5 independent experiments. ＊＊ , P ＜0.01 vs. WT, † , P ＜0.05, †† , P ＜0.01 vs. KD. F, Successful overexpression of PGK1 protein by transfection of sPLA 2 -V KD RAW264.7 cells with a plasmid vector encoding Pgk1 . Values were normalized to that of WT (=1). G, Schematic representation of a potential mechanism for sPLA 2 -V mediated endocytosis of AcLDL in RAW264.7 cells.

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Translocation Assay, Expressing, Flow Cytometry, Control, Western Blot, Transfection, Plasmid Preparation, Over Expression

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, Ratio of c-Src phosphorylation at the active site (Y416) relative to total c-Src in response to AcLDL was reduced in sPLA 2 -V KD cells compared with that for sPLA 2 -V WT cells. Data are expressed as values relative to the value of sPLA 2 -V WT cells at baseline (incubation time 0) (=1). n =5 in each experiment. ＊ , P ＜0.05, vs. WT. B, Representative immunoblots for panel A. C and D, Overexpression of c-Src after transfection with an expression vector encoding the c-Src gene reversed an impairment of actin polymerization (C) and transport of internalized AcLDL conjugated with pHrodo to lysosomes (D) in sPLA 2 -V KD RAW264.7 cells. Details of the methods are described in the text. n =5 in each experiment. ＊ , P ＜0.05, ＊＊ , P ＜0.01 vs. WT, †† , P ＜0.01 vs KD. E, Immunoblotting showing successful expression of c-Src protein driven by an expression vector used to transfect sPLA 2 -V KD RAW264.7 cells. Values were normalized to that of WT (=1). F, siRNA (#1 and #2)-mediated reduction of PGK1 suppressed c-Src phosphorylation at Y416 in response to AcLDL in sPLA 2 -V WT RAW264.7 cells. G, Representative immunoblots for panel F. H, siRNA-mediated reduction of Beclin1 suppressed c-Src phosphorylation at Y416 in response to AcLDL in sPLA 2 -V WT RAW264.7 cells. I, Representative immunoblots for panel H. Values in panels F and H were normalized to that of control siRNA at baseline (incubation time 0)(=1). n =5 in each experiment, ＊ , P ＜0.05, ＊＊ , P ＜0.01 vs. control siRNA.

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Phospho-proteomics, Translocation Assay, Incubation, Western Blot, Over Expression, Transfection, Expressing, Plasmid Preparation, Control

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A and B, siRNA-mediated reduction of expression of sPLA 2 -IID, -IIE, or -XIIA did not change translocation of pHrodo-conjugated AcLDL to lysosomes (A) and expression of Pgk1 mRNA at baseline (B). MFI indicates mean fluorescence intensity. n =5 in each experiment. Values in B were normalized to that of control siRNA (=1). C, Reduction of expression of sPLA 2 -IID, -IIE, or -XIIA mRNA by their respective siRNAs (#1 and #2). n =5 in each experiment. Values were normalized to that of control siRNA (=1).

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Knockdown, Translocation Assay, Expressing, Fluorescence, Control

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Group V Secretory Phospholipase A 2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

doi: 10.5551/jat.62216

Figure Lengend Snippet: A, B, and C, sPLA 2 -V KO peritoneal macrophages had impaired specific internalization (A) and degradation (B) of 125 I AcLDL but a similar degree of cell surface-specific binding (C) compared to sPLA 2 -V WT peritoneal macrophages. D and E, sPLA 2 -V KO peritoneal macrophages had decreased amounts of actin polymerization (D) and translocation of internalized AcLDL conjugated with pHrodo (E) compared with sPLA 2 -V WT macrophages. F, G, and H, PGK1 expression at baseline (F), Beclin1 phosphorylation at S30 (G), and c-Src phosphorylation at Y416 (H) were decreased in peritoneal macrophages from sPLA 2 -V KO mice compared with those from sPLA 2 -V WT mice. Values in panels F, G, and H were normalized to that of WT (incubation time 0 in panels G and H) (=1). Each bar represents the mean±SEM of 5 independent experiments. ＊ , P ＜0.05, ＊＊ , P ＜0.01 vs. WT. I, Representative immunoblots in panels of G and H.

Article Snippet: A mouse PGK1 expression vector (accession no. NM_008828) was purchased from Origene Technologies.

Techniques: Knock-Out, Binding Assay, Translocation Assay, Expressing, Phospho-proteomics, Incubation, Western Blot

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 1. PGK1 deficiency attenuates systemic and peritoneal inflammation and impairs NLRP3 inflammasome activation (A) Survival curves of PGK1 fl/fl , PGK1 mKO mice (n = 10 mice/group) after intraperitoneal LPS injection (8 mg/kg). (B and C) Serum IL-1β (B) and TNF-α (C) levels at 6 h post-LPS injection (n = 5 per group). (D) Flow cytometry analysis of peritoneal CD11b + Ly6G + cells (neutrophils) from PGK1 fl/fl , PGK1 mko mice at 12 h after intraperitoneal injection with alum (n = 5 per group). (E) Immunoblot of LPS-primed BMDMs (PGK1 fl/fl , PGK1 mKO ) treated with nigericin; cell lysates (Lysate) and supernatants (SN) were probed for NLRP3 pathway components. (F–H) ELISA quantification of IL-1β (F), TNF-α (G), and LDH release (H) in supernatants. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA with Tukey’s test). Mice were randomly assigned by sex. Data are representative of at least three independent experiments.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Activation Assay, Injection, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 2. PGK1 interacts with NLRP3 via phosphorylation at S271 (A) Co-immunoprecipitation (coIP) of HA-PGK1 and FLAG-NLRP3 in HEK293T lysates with the indicated antibodies. (B) LPS-primed BMDMs were unstimulated or stimulated with nigericin for 30 min. Cell lysates were IP and immunoblotted with the indicated antibodies. (C) Immunofluorescence of PGK1 (green) and NLRP3 (red) in BMDMs ± nigericin. Scale bar: 10 μm. (D–F) Domain mapping of PGK1-NLRP3 interaction using truncation mutants. (G) Evolutionary conservation of PGK1(267 − 276). (H and I) CoIP analysis of PGK1 phosphorylation mutants and NLRP3 binding. PGK1(3K/R) refers to lysine-to-arginine mutations at residues K256, K258, and K260 in PGK1(267–276). (J) Prediction of the potential key amino acids on NLRP3 that determine the interaction with PGK1 phosphorylated with 271 serine.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Phospho-proteomics, Immunoprecipitation, Immunofluorescence, Binding Assay

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 3. PGK1 S271 phosphorylation enhances NLRP3 inflammasome activity (A) Immunoblot of LPS/nigericin-treated WT and PGK1 S271A/S271A BMDMs. (B) LPS stimulated BMDMs for 0, 8, and 16 h, and they were analyzed by immunofluorescence as indicated (yellow arrows). Scale bar, 10 μm. (C) ASC oligomerization in WT and PGK1 S271A/S271A BMDMs. (D) Quantification of ASC specks (white arrows). Scale bar: 20 μm. (E–H) LPS-primed BMDMs (WT, PGK1 S271A/S271A ) treated with LPS for 4 h and then stimulated with nigericin or MCC950 (an NLRP3 inhibitor, 10 μМ), immunoblot analysis of cell lysates (Lysate) and culture supernatants (SN) as indicated, and ELISA assay for LDH release (F), IL-1β secretion (G), and TNF-α secretion (H) in supernatants. (I) PGK1 peptide (10aa WT and 10aa S271D) conjugated to cell-penetrating TAT peptide. (J) LPS-primed BMDMs treated with LPS/nigericin, with peptides (10aa WT or 10aa S271D), immunoblot analysis of cell lysates (Lysate), and culture supernatants (SN) as indicated. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA with Tukey’s test). ns, not significant. Data are representative of at least three in- dependent experiments.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 4. CK2-mediated PGK1 S271 phosphorylation drives NLRP3 activation (A) The sequence of amino acids 267 to 276 of PGK1 is conserved in multiple species and contains the recognition motif of CK2. (B) In vitro kinase assay of Myc-CK2 with GST-PGK1/S271A. Myc-CK2 was immunoprecipitated with anti-Myc from 293T cells transfected with Myc-CK2 incubated with 20 μL purified GST-PGK1 and GST-PGK1S271A protein, 50 μM ATP, 10× kinase assay buffer, and 1× protease inhibitor cocktail at 30 ◦ C for 30 min, analyzed by immunoblotting with antibodies as indicated. (C) HEK293T cells were transfected with the indicated vectors. Samples were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting as indicated. (D) HEK293T cells were transfected with the indicated vectors and treated with or without TBB (5 μM). Samples were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting as indicated. (E) LPS-primed WT and PGK1 S271A/S271A BMDMs were stimulated with nigericin (45 min) ± TBB (5 μM); immunoblot analysis of cell lysates (Lysate) for indicated proteins. (F–H) ELISA quantification of IL-1β (F), TNF-α (G), and LDH release (H) in supernatants. (I and J) BMDMs from WT and PGK1 S271A/S271A mice were treated with LPS for 4 and 16 h. The extracellular acidification and oxygen consumption rates were measured with the XF24 Seahorse Analyzer. The bottom bar charts display the maximum glycolytic capacity and maximum respiratory capacity, respectively. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA with Tukey’s test). ns, not significant. Data are representative of at least three in- dependent experiments.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Phospho-proteomics, Activation Assay, Sequencing, In Vitro, Kinase Assay, Immunoprecipitation, Transfection, Incubation, Purification, Protease Inhibitor, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 5. PGK1 phosphorylates NLRP3 at S448/449 to modulate ubiquitination (A) CoIP of HA-PGK1 with FLAG-NLRP3 in HEK293T lysates; immunoblotting with indicated antibodies. (B) Sequence alignment of NLRP3 residues 436–457 and 1,010–1,024 across various species. (C) Ubiquitination assays in HEK293T cells expressing Myc-NLRP3, FLAG-NLRP3 (WT, S448A/449A, S1016A), and HA-ubiquitin; lysates immunoprecipitated with anti-FLAG. (D) Ubiquitination analysis of FLAG-NLRP3 (WT, S448A, S449A) co-expressed with Myc-ubiquitin; immunoblotting with indicated antibodies. (E) CoIP of HA-PGK1 with FLAG-NLRP3 (WT, S448A/449A) in HEK293T cells. Samples were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting as indicated. (F) LPS-primed PMs (WT, NLRP3S448A/449A, NLRP3S448D/449D) treated LPS for 4 h and then stimulated with nigericin. Immunoblot analysis of cell lysates (Lysate) and culture supernatants (SN) as indicated. (G–I) ELISA quantification of LDH (G), IL-1β (H), and TNF-α (I) in supernatants. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA with Tukey’s test). Data are representative of at least three independent experiments.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Ubiquitin Proteomics, Western Blot, Sequencing, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 6. PGK1 promotes NLRP3 deubiquitination via USP14 (A) Ubiquitination analysis of NLRP3 in HEK293T cells transfected with indicated constructs; IP with anti-FLAG antibody and immunoblotting as shown. (B) BMDMs primed with LPS (200 ng/mL, 4 h) and treated with WP1130 (0, 2.5, 5 μM, 30 min). Cell lysates were immunoprecipitated and analyzed by immu- noblotting as indicated. (C) CoIP of Myc-NLRP3, FLAG-USP14, and HA-PGK1 in HEK293T lysates using anti-FLAG antibody and analyzed by immunoblotting as indicated. (D) HEK293T cells were transfected with the indicated vectors, and samples were immunoprecipitated with the anti-HA antibody and analyzed by immunoblotting as indicated. (E) PGK1 fl/fl and PGK1 mKO BMDMs were primed with LPS 4 h, and cell lysates were immunoprecipitated with the anti-NLRP3 and analyzed by immunoblotting as indicated. (F) Predicted interaction interface between USP14 and phosphorylated NLRP3 (p-S448/p-S449) based on computational modeling. (G) HEK293T cells were transfected with the indicated vectors. Samples were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting as indicated. (H) HEK293T cells were transfected with the indicated vectors treated with vehicle or TBB. Samples were immunoprecipitated with the anti-Myc antibody and analyzed by immunoblotting as indicated. (I) HEK293T cells were transfected with the indicated vectors. Samples were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting as indicated.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Ubiquitin Proteomics, Transfection, Construct, Western Blot, Immunoprecipitation

Journal: Cell reports

Article Title: PGK1 phosphorylates NLRP3 and mediates inflammasome activation independent of its glycolytic activity.

doi: 10.1016/j.celrep.2025.115785

Figure Lengend Snippet: Figure 7. PGK1 S271 phosphorylation increases NLRP3 inflammasome activity (A) Survival of WT and WT and PGK1 S271A/S271A mice (n = 5–10 per group) after intraperitoneal injection of MCC950 (50 mg/kg), WP1130 (40 mg/kg), or TBB (60 mg/kg), followed by LPS (8 mg/kg) for 72 h. (B and C) Serum IL-1β (B) and TNF-α (C) levels in WT and PGK1 S271A/S271A mice 6 h post-LPS injection (n = 3 per group). (D) Flow cytometry analysis of peritoneal CD11b + Ly6G + cells (neutrophils) 12 h after alum challenge (n = 3 per group). (E and F) ELISA quantification of IL-1β and TNF-α in serum (E) and ascites (F). Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA with Tukey’s test). ns, not significant. Mice were randomly assigned by sex. Data are representative of at least three independent experiments.

Article Snippet: PGK1 flox/flox mice were obtained from Cyagen.

Techniques: Phospho-proteomics, Activity Assay, Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Molecular Biology of the Cell

Article Title: Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

doi: 10.1091/mbc.E14-04-0870

Figure Lengend Snippet: Cell migration driven by localized Gi protein inhibition. (A) Image sequence of a live RAW 264.7 cell transiently transfected with CRY2-mCh-RGS4Δ, CIBN-CaaX, PH(Akt)-Venus, and CXCR4. Local OA was applied to generate a CRY2-mCh-RGS4Δ gradient before uniform addition of SDF-1α. Scale bar, 10 μm. (B) Negative control expressing CRY2-mCh-PGK1 instead of CRY2-mCh-RGS4Δ. (C, D) The t -stacks corresponding to the data in A and B. Localization of the RGS construct, but not the PGK construct, results in a PIP3 gradient, directional cell protrusions, and migration. White boxes correspond to OA regions. Yellow boxes show regions selected for generating the corresponding t -stacks.

Article Snippet: A PCR product of PGK1 (38071; Addgene) was inserted into the Kpn I and Xba I sites of CRY2PHR-mCh to create CRY2-mCh-PGK1.

Techniques: Migration, Inhibition, Sequencing, Transfection, Negative Control, Expressing, Construct