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: 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: 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

Journal: PLOS Genetics

Article Title: Mms4 chromosomal association reveals functional relationships between meiotic crossover pathways in budding yeast

doi: 10.1371/journal.pgen.1012097

Figure Lengend Snippet: A) Western blot analysis of Mms4-9xMyc expression from 0 to 9 hours after meiotic induction. Pgk1 serves as a loading control. B) Mms4-9xMyc ChIP using the anti-Myc antibody in synchronized wild-type meiotic cultures. Lanes 1, 3, and 5 represent the Mms4-9xMyc strain; lanes 2, 4, and 6 represent the untagged wild-type strain. M: molecular weight marker. C) Calibrated ChIP-seq profile showing Mms4-9xMyc binding on chromosome III at 3,4 and 5 hours post meiotic induction. Red1 and Spo11 data are from and , respectively. The black circle marks the centromere. Dotted lines indicate centromeric region, axis region, DSB hotspot ( BUD23 ), and DSB coldspot ( YCR093W ). D) Density plot of Mms4 ChIP-Seq read counts at Mms4 peak locations at 3h, 4h, and 5h post meiotic induction. The X-axis represents Mms4 binding strength, measured by the number of Mms4 reads in 10 bp bins at each peak, while the Y-axis indicates the probability density for a given number of Mms4 reads in the genomic bins.

Article Snippet: The following primary antibody dilutions were used: mouse anti-Myc antibody (Sigma, M4439, 1:1000); mouse anti-Pgk1 antibody (Novus Biologicals, NBP1–33685, 1:30,000).

Techniques: Western Blot, Expressing, Control, Molecular Weight, Marker, ChIP-sequencing, Binding Assay

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: PGK1 acts as an Hsp90 co-chaperone specifically regulating glycogen synthase kinase-3 (GSK3β) expression. (A) The interaction of GSK3β with Hsp90 and PGK1 in MCF-7ADR cells. GSK3β was immunoprecipitated using an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (B) The interaction of PGK1 with chaperones, co-chaperones, and client proteins. MCF-7ADR cells were transfected with 2 μg PGK1-Flag plasmid for 48 h. PGK1-Flag was immunoprecipitated using an anti-flag antibody. Immunoblotting analyses were performed with the indicated antibodies. (C) The interaction of Hsp90 with GSK3β and PGK1 in MCF-7ADR cells. Hsp90 was immunoprecipitated from cell lysates using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (D) The interaction of Hsp90 with GSK3β and PGK1 in clinical breast cancer tissues of 3 patients. Hsp90 was immunoprecipitated from lysates using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (E, G) PGK1 depletion (E) or (G) overexpression on client protein expression of Hsp90. MCF-7ADR cells were transfected with PGK1 siRNA (E) or PGK1-Flag (G) for 48 h. Immunoblotting analyses were performed with the indicated antibodies. (F) The effect of PGK1 knockdown on GSK3β expression in the indicated cells. Immunoblotting analyses were performed with the indicated antibodies. (H) The effect of PGK1 knockdown on the interaction between Hsp90 and GSK3β. MCF-7ADR cells were transfected with PGK1 siRNA. After 36 h, cells were treated with 20 μM MG132 for 12 h, and then subjected to IP using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (I, J) The effect of HOP (I) or CDC37 (J) knockdown on GSK3β expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblotting analyses were performed with the indicated antibodies. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-PGK1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Over Expression, Knockdown

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: PGK1 binds to the C-terminus of Hsp90 in the “closed” state to promote the interaction between glycogen synthase kinase-3 GSK3β and Hsp90. (A) Constructed 293T cells stably expressing flag-tagged Hsp90α full-length (WT), N-, M-, or C- domains. Empty vector was used as a control. (B) The interaction of different Hsp90α domains with PGK1 and GSK3β. Different domains of Flag-Hsp90α were immunoprecipitated using an anti-Flag antibody. Immunoblotting analyses were performed with the indicated antibodies. (C, D) The interaction of PGK1 with the C-terminus (C) or M-domain (D) of Hsp90α in vitro . A total of 5 μg GST-C/M-Hsp90α and 2.5 μg His-PGK1 were incubated at 30 °C for 1 h. Immunoprecipitation (IP) was performed with an anti-GST antibody. Immunoblotting analyses were performed with the indicated antibodies. (E) The interaction of PGK1 with GSK3β in vitro . A total of 2.5 μg His-GSK3β and 2.5 μg His-PGK1 were incubated at 30 °C for 1 h. IP was performed with an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (F) PGK1 regulates the interaction between C-terminal Hsp90α and GSK3β in vitro . A total of 5 μg GST-C-Hsp90α and 2.5 μg His-GSK3β were incubated with or without 2.5 μg His-PGK1. IP was performed with an anti-GST antibody. Immunoblotting analyses were performed with the indicated antibodies. (G) The interaction of PGK1 with different conformations of Hsp90α. Flag-tagged Hsp90α wild type, D93A, or E47A mutant was stably expressed in 293T cells. Flag-tagged Hsp90 were immunoprecipitated. Immunoblotting analyses were performed with the indicated antibodies. (H) The binding mode of PGK1 with the “closed” conformation of Hsp90β. The N-terminal and C-terminal of Hsp90β are colored in blue and magenta, respectively, while the PGK1 is shown in green. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-PGK1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Construct, Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, In Vitro, Incubation, Mutagenesis, Binding Assay

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: Hsp90 inhibitors targeting different domains of Hsp90 exhibit distinct inhibitory effects on BCSCs. (A) Flow cytometry (FCM) sorting of CD44 + CD24 −/low cells (BCSCs) and CD44 + CD24 + cells (non-BCSCs) from MCF-7ADR cells. (B) Immunofluorescence analysis of BCSCs and non-BCSCs. CD44 + CD24 −/low cells and CD44 + CD24 + cells were stained with an anti-GSK3β pS9 antibody and 4′,6-diamidino-2-phenylindole. (C) Immunoblotting analysis of BCSCs and non-BCSCs were performed with the indicated antibodies. (D) The effect of glycogen synthase kinase-3 knockdown on ALDH1A1 expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblot analyses were performed with the indicated antibodies. (E) The effect of PGK1 knockdown on ALDH1A1 expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblot analyses were performed with the indicated antibodies. (F, G) The effects of HDN-1 (F) and 17-AAG (G) on the proliferation of MCF-7ADR cells. The cells were treated with HDN-1 or 17-AAG for 48 h. Cell proliferation was determined using the MTT assay. (H-K) The effects of HDN-1 (H, I) and 17-AAG (J, K) on ALDH1A1 expression in MCF-7ADR cells. Cells were treated with HDN-1 or 17-AAG for 48 h. Immunoblot analyses of BCSCs and non-BCSCs were performed with the indicated antibodies. (L-O) The effects of HDN-1 (L, M) and 17-AAG (N, O) on mammosphere formation of MCF-7ADR cells. A total of 2,000 MCF-7ADR cells per well were cultured in serum-free medium and then treated with different concentrations of HDN-1 or 17-AAG. After 7 days, images were captured using a light microscope with a camera, and the number of mammospheres was quantified. The scale bar is 10.0 μm. * P < 0.05; ** P < 0.01 vs . dimethyl sulfoxide. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-PGK1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Western Blot, Knockdown, Expressing, Transfection, MTT Assay, Cell Culture, Light Microscopy

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: 17-AAG and HDN-1 have distinct effects on Hsp90-PGK1 interaction and subsequent expressions of GSK3β and β-catenin. (A, C) The effect of HDN-1 in a dose-dependent (A) or time-course (C) treatment on the expressions of proteins related to Akt-GSK3β-β-catenin signaling in MCF-7ADR cells for 24 h. (B) The effect of HDN-1 on the phosphorylation level of β-catenin at Ser 45 in MCF-7ADR cells. (D) The effect of HDN-1 on polyubiquitination of glycogen synthase kinase-3 (GSK3β). MCF-7ADR cells were treated with HDN-1 for 12 h, followed by co-treatment with MG132 for 12 h. GSK3β was immunoprecipitated from cell lysates using an anti-GSK3β antibody. (E, F) The effect of 17-AAG in a dose-dependent (E) or time-course (F) treatment on the expression of Akt, GSK3β, p-β-catenin at Ser45, and β-catenin in MCF-7ADR cells. (G, H) The effects of HDN-1 (G) and 17-AAG (H) on the expressions of Akt, GSK3β, and β-catenin. The 293T cells were treated with HDN-1 or 17-AAG for 24 h. (I, J) The effects of HDN-1 (I) and 17-AAG (J) on the binding of Hsp90 to Akt and GSK3β. MCF-7ADR cells were treated with HDN-1 or 17-AAG. Hsp90 was immunoprecipitated from cell lysates using an anti-Hsp90 antibody. (K, L) The effects of HDN-1 (K) and 17-AAG (L) on PGK1 expression. (M) The effects of HDN-1 and 17-AAG on the formation of the Hsp90α-PGK1-GSK3β complex in vitro . Hsp90α, His-PGK1, and His-GSK3β were incubated, followed by treatment with 1 μM HDN-1 or 17-AAG at 4 °C for 1 h. Immunoprecipitation was performed using an anti-Hsp90 antibody. (N) The effects of HDN-1 and 17-AAG on the interaction between PGK1 and the “closed” conformational Hsp90α. The 293T cells expressing Flag-tagged Hsp90-E47A were treated with HDN-1 or 17-AAG for 24 h. Flag-Hsp90-E47A was then immunoprecipitated from cell lysates. Immunoblotting analyses were performed with the indicated antibodies. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-PGK1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Immunoprecipitation, Expressing, Binding Assay, In Vitro, Incubation, Western Blot

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: Schematic of the distinct effects of HND-1 and 17-AAG on stemness of breast cancer by affecting Hsp90 regulation of glycogen synthase kinase-3 (GSK3β). The 17-AAG and HDN-1 had distinct effects on Hsp90-PGK1 interaction, resulting in different changes and the instability of GSK3β. They all inhibited AKT expression and its phosphorylation. HDN-1 significantly inhibited the Wnt-β-catenin cascade, yet 17-AAG did not affect the signaling pathway, leading to different effects on the stemness of breast cancer.

Article Snippet: Anti-PGK1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Phospho-proteomics