Journal: Research

Article Title: PGK1 Lactylation-Driven Self-Reinforcing Loop Orchestrates Glycolytic Reprogramming in FSP1 + Macrophages in Liver Fibrosis

doi: 10.34133/research.1177

Figure Lengend Snippet: FSP1-induced lactate accumulation drives PGK1 lactylation at K353 by KAT2B lactyltransferase. (A) Relationship of FIB4 and serum lactate from cirrhosis patients ( n = 102). (B) Fsp1f/f and LysM cre Fsp1 f/f mice were subjected to CCl 4 -induced hepatic fibrosis modeling ( n = 3 per group). Liver tissues were collected, and whole protein lysates were prepared. Immunoprecipitation (IP) was performed using pan-lactylated lysine antibodies, followed by mass spectrometry (MS) analysis to identify lactylated proteins. The graph depicts the number of proteins with significantly altered lactylation levels between fibrotic Fsp1f/f and LysM cre Fsp1 f/f livers, determined by quantitative MS-based proteomics. (C) Pathway enrichment of DEPs using KEGG database defined as in (B). (D) Heatmap depicting lactylation levels of lysine residues in glycolysis-related proteins with statistically significant changes. (E) Detecting lactylation of the proteins identified in (D) by Western blot using IP sample as indicated. n = 3 technical replicates per group. (F and G) PMA-differentiated THP1 (F) cells and BMDMs (G) were treated for 24 h with NALA or LDHi (Oxamate [Oxa]) and induced to an M1 phenotype. Cell lysates were immunoprecipitated with an anti-PGK1 antibody, followed by immunoblot analysis using anti-Pan-Lac or anti-PGK1 antibodies. n = 3 technical replicates per group. (H) HEK293T cells transfected with Flag-tagged PGK1 plasmid were treated with NALA or Oxa. IP was performed to detect the lactylation modification levels of PGK1. n = 3 technical replicates per group. (I) HEK293T cells were pretreated with 20 mM MCT1/4i for 12 h prior to transfection with a Flag-tagged PGK1 plasmid. Detecting lactylation of PGK1 using IP samples as indicated. n = 3 technical replicates per group. (J to L) Detection of PGK1 lactylation in Fsp1f/f and LysM cre Fsp1 f/f fibrotic liver tissues induced by CCl 4 injection (J), MCD diet (K), and BDL (L) using the indicated IP samples. IgG serves as the control antibody group. n = 3 technical replicates per group. (M and N) The lactylation of PGK1 was measured in IP samples from PMA-differentiated THP1 (M) and BMDMs (N) that were transfected with siFsp1 and polarized to the M1 phenotype. n = 3 technical replicates per group. (O) Illustration of PGK1 K353 lactylation identified by MS. (P) PGK1 lactylation assay was performed in HEK293T cells transfected with Flag-tagged WT-PGK1 or K353R-PGK1 plasmids, followed by treatment with 25 mM NALA for 24 h. n = 3 technical replicates per group. (Q) BMDMs were transduced with lentiviral vectors encoding WT PGK1 or the K353R mutant to establish stable overexpression, followed by induction of M1 macrophage polarization. Lactylation of PGK1 was subsequently assessed in IP lysates using Western blot analysis with the indicated antibodies. n = 3 technical replicates per group. (R) Screening the “writer(s)” of PGK1 lactylation by transfecting combined Flag-tagged PGK1 and acetyltransferase as indicated. (S) The cofactor binding pockets of KAT2B (PDB ID: 4NSQ) are shown in complex with acetyl-CoA (top panel) and lactyl-CoA (bottom panel). KAT2B is depicted as a cartoon model, and the transferable moieties in acetyl-CoA or lactyl-CoA are marked with red circles. (T and U) Co-IP showing interactions between KAT2B and PGK1 in BMDMs. n = 3 technical replicates per group. (V) Lactylation detection of PGK1 was performed via IP in HEK293T cells that had been transfected with Flag-tagged PGK1 plasmid and treated with siRNA targeting KAT2B ( siKat2b ), as evidenced by Western blot analysis. n = 3 technical replicates per group. (W) Detecting PGK1 lactylation using an in vitro lactylation assay on the indicated samples. n = 3 technical replicates per group. (X) Detecting lactylation of PGK1 in BMDMs treated with NAM using IP samples as indicated. n = 3 technical replicates per group.

Article Snippet: Glutathione S -transferase-fused PGK1 protein was coincubated with HA-tagged KAT2B protein purified from HEK293T cells in reaction buffer (50 mM HEPES, pH 7.8, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM dithiothreitol), supplemented with 20 mM lactyl-CoA (HY-141540, MCE, USA) [ ].

Techniques: Immunoprecipitation, Mass Spectrometry, qMS Based, Western Blot, Transfection, Plasmid Preparation, Modification, Injection, Control, Transduction, Mutagenesis, Over Expression, Binding Assay, Co-Immunoprecipitation Assay, In Vitro

Journal: Research

Article Title: PGK1 Lactylation-Driven Self-Reinforcing Loop Orchestrates Glycolytic Reprogramming in FSP1 + Macrophages in Liver Fibrosis

doi: 10.34133/research.1177

Figure Lengend Snippet: Glycolysis/PGK1 lactylation forms a positive feedback loop in FSP1 + macrophages of mice with hepatic fibrosis. (A) Enzymatic activity of PGK1 was assessed in HEK293T cells transfected with Flag-tagged PGK1 WT or K353R point mutant construct, followed by treatment with NALA. n = 3 per group. (B) The enzymatic activity of PGK1 was measured in liver tissues isolated from Fsp1 f/f and LysM cre Fsp1 f/f mice with liver fibrosis induced by CCl 4 injection, MCD diet, or BDL. n = 6 per group. (C) The enzymatic activity of PGK1 was measured in PMA-differentiated THP-1 and BMDMs with the indicated treatment. n = 3 biological replicates. (D) BMDMs and THP1 transfected with siFsp1 or siCtrl were polarized with M1-inducing stimuli, followed by triple staining with anti-PGK1 antibody (green), MitoTracker (red), and DAPI (blue). Scale bar, 50 μm. n = 3 biological replicates. (E) Representative immunofluorescence images of HEK293T cells were transfected with Flag-tagged PGK1 WT or K353R mutant construct, under untreated (NC) or stimulated with NALA (50 mM, 24 h) conditions. Cells were costained with anti-Flag antibody (green, PGK1 localization) and MitoTracker Red (mitochondrial marker). Nuclear counterstaining was performed with DAPI (blue). Scale bar, 50 μm. n = 3 biological replicates. (F) PDHK1 expression was analyzed by IP in HEK293T cells expressing Flag-PGK1 WT or its K353R point mutant construct. n = 3 biological replicates. (G) BMDMs transfected with siFsp1 or siCtrl were polarized with M1-inducing cytokines. Whole-cell lysates were subjected to IP using anti-PGK1 antibody, followed by immunoblotting for PDHK1. n = 3 biological replicates. (H) Immunoblotting analysis of PDHK1 and PDH phosphorylation in mitochondria. Mitochondrial fractions were prepared from BMDMs and immunoblotted with indicated antibodies. n = 3 biological replicates. (I) PDHK1 and PDH phosphorylation was detected in mitochondria by immunoblotting analysis. Mitochondrial fractions were prepared from HEK293T cells receiving Flag-tagged PGK1 WT or K353R point mutant construct. n = 3 biological replicates. (J) PGK1 phosphorylation was detected in BMDMs with the indicated treatment. n = 3 biological replicates. (K) PGK1 phosphorylation was detected in HEK293T cells transfected with Flag-tagged PGK1 WT or K353R point mutant construct, and followed by NALA treatment. n = 3 biological replicates. (L) BMDMs transfected with lentiviruses overexpressing WT PGK1 or the K353R mutant were polarized to M1 phenotype. ERCR and OCR were measured to assess glycolysis and mitochondrial oxidative phosphorylation, respectively. n = 5 to 6 per group. Data were presented as mean ± SEM; Statistical significance was determined by unpaired Student t test (A and B) or one-way ANOVA (C). * P < 0.05, *** P < 0.001, **** P < 0.0001. ns, no significant difference.

Article Snippet: Glutathione S -transferase-fused PGK1 protein was coincubated with HA-tagged KAT2B protein purified from HEK293T cells in reaction buffer (50 mM HEPES, pH 7.8, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM dithiothreitol), supplemented with 20 mM lactyl-CoA (HY-141540, MCE, USA) [ ].

Techniques: Activity Assay, Transfection, Mutagenesis, Construct, Isolation, Injection, Staining, Immunofluorescence, Marker, Expressing, Western Blot, Phospho-proteomics

Journal: Research

Article Title: PGK1 Lactylation-Driven Self-Reinforcing Loop Orchestrates Glycolytic Reprogramming in FSP1 + Macrophages in Liver Fibrosis

doi: 10.34133/research.1177

Figure Lengend Snippet: PGK1 inhibition prevents the progression of liver fibrosis. (A) The MFIs of MHC-II were detected in BMDMs subjected to NG52 (a PGK1 inhibitor, 25 μM, 24 h) treatments after M1 induction, as determined by flow cytometry. n = 3 biological replicates. (B) The enzymatic activity of PGK1 was assessed in the indicated groups. n = 3 biological replicates. (C) THP1-derived macrophages and BMDMs were polarized to M1 phenotype, followed by treatment with NG52 or vehicle control. Cell lysates were subjected to immunoblotting with anti-K353la-PGK1 antibody for site-specific lactylation detection and anti-total PGK1 antibody for loading control for normalization. n = 3 per group. (D) Immunoblotting analysis of iNOS and PDH phosphorylation in M1-polarized THP1 and BMDMs treated with NG52 or vehicle control. n = 3 per group. (E) mRNA expression of α-SMA , Col1α1 , Fibronectin , and Timp1 was quantified in 3 murine models of liver fibrosis induced by CCl 4 injection, MCD diet, or BDL with NG52 or vehicle control. n = 6 per group. (F) mRNA expression of Tnf-α , Il-1β , and Il-10 was quantified in the mice. (G) Serum TNF-α, IL-1β, and IL-10 were measured by ELISA in the indicated groups. n = 6 per group. (H) The degree of fibrosis was evaluated by H&E, Masson’s trichrome, Sirius Red, collagen I, and α-SMA immunohistochemical staining, and the degree of inflammation was evaluated by Ly6G immunohistochemical staining in the indicated groups. Scale bars, 50 μm. (I) Protein expression of collagen I, α-SMA, and iNOs was analyzed by Western blot analysis in liver samples. Data were presented as mean ± SEM. Data were analyzed by an unpaired Student t test, with statistical significance set at * P < 0.05 and ** P < 0.01.

Article Snippet: Glutathione S -transferase-fused PGK1 protein was coincubated with HA-tagged KAT2B protein purified from HEK293T cells in reaction buffer (50 mM HEPES, pH 7.8, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM dithiothreitol), supplemented with 20 mM lactyl-CoA (HY-141540, MCE, USA) [ ].

Techniques: Inhibition, Flow Cytometry, Activity Assay, Derivative Assay, Control, Western Blot, Phospho-proteomics, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Research

Article Title: PGK1 Lactylation-Driven Self-Reinforcing Loop Orchestrates Glycolytic Reprogramming in FSP1 + Macrophages in Liver Fibrosis

doi: 10.34133/research.1177

Figure Lengend Snippet: Inhibiting PGK1 K353 lactylation with a peptidic inhibitor disrupts the glycolysis/PGK1 lactylation positive feedback loop in FSP1 + macrophages. (A) Liver tissues were collected from 24 patients with liver cirrhosis and 12 patients undergoing hemangioma surgery for Western blotting analysis. (B) Clinical liver fibrosis tissues were subjected to immunohistochemistry staining, and the representative staining results of samples with PGK1 K353la levels were shown. Scale bar, 50 μm. n = 20 for cirrhosis samples and n = 8 for the controls. (C) PGK1 lactylation was analyzed in M1-polarized THP1 cells and BMDMs, followed by treatment with K353-peptide (50 μM) or K353R-peptide (50 μM) as control for an additional 24 h. Western blot analysis was performed to detect K353 lactylation and PGK1 expression. n = 3 biological replicates. (D) PGK1, PDHK1, and PDH phosphorylation was detected by immunoblotting analysis. n = 3 biological replicates. (E) M1-polarized BMDMs were treated with K353-peptide or K353R-peptide, followed by staining with anti-PGK1 antibody and MitoTracker. Nuclei were counterstained with DAPI. n = 3 biological replicates. (F) The enzymatic activity of PGK1 was assessed in the indicated groups. n = 3 biological replicates. (G) Immunoblotting analysis of iNOS was assessed in the indicated groups. n = 3 biological replicates. (H) The MFIs of MHC-II were detected in BMDMs subjected to the indicated treatments, as determined by flow cytometry. n = 3 biological replicates. (I) The ERCR and OCR were determined in M1 polarized-BMDMs receiving K353R-pe or K353-pe. n = 3 biological replicates. (J) Sirius Red staining and collagen I IF staining were used to assess fibrosis severity, while α-SMA IF staining evaluated HSC activation. Scale bars, 50 μm. n = 3 biological replicates. Data were presented as mean ± SEM. Statistical significance was determined by unpaired Student t test (E) or one-way ANOVA (H). * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Glutathione S -transferase-fused PGK1 protein was coincubated with HA-tagged KAT2B protein purified from HEK293T cells in reaction buffer (50 mM HEPES, pH 7.8, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM dithiothreitol), supplemented with 20 mM lactyl-CoA (HY-141540, MCE, USA) [ ].

Techniques: Western Blot, Immunohistochemistry, Staining, Control, Expressing, Phospho-proteomics, Activity Assay, Flow Cytometry, Activation Assay

Journal: Research

Article Title: PGK1 Lactylation-Driven Self-Reinforcing Loop Orchestrates Glycolytic Reprogramming in FSP1 + Macrophages in Liver Fibrosis

doi: 10.34133/research.1177

Figure Lengend Snippet: Targeting lactylation of PGK1 K353 via a peptidic inhibitor alleviates the severity of liver fibrosis. (A) PGK K353la was detected in fibrotic mice using 3 distinct models: CCl 4 injection, MCD diet, and BDL. Mice were treated with either K353-peptide (K353-pe) or K353R-peptide (K353R-pe, control). n = 6 per group. (B) Liver tissue lactate and PGK1 activity were determined in fibrotic mice with the indicated treatment. n = 6 per group. (C) mRNA expression of α-SMA , Col1a1 , and Fibronectin was quantified in the indicated groups. n = 6 biological replicates per group. (D) mRNA expression of Tnf-α , Il-1β , and Il-10 was quantified in the indicated groups. n = 6 per group. (E) Serum TNF-α, IL-1β, and IL-10 were measured by ELISA in the indicated groups. n = 6 per group. (F) The degree of fibrosis was assessed by H&E, Masson’s trichrome, Sirius Red staining, collagen I, and α-SMA immunohistochemical staining, and the degree of inflammation was evaluated by Ly6G immunohistochemical staining in the indicated groups. n = 6 per group. Scale bars, 50 μm. (G) Protein expression of collagen I, α-SMA, and iNOS was analyzed in liver samples by Western blot analysis. n = 3 technical replicates per group. Data were presented as mean ± SEM; Statistical significance was determined by unpaired Student t test. * P < 0.05, ** P < 0.01.

Article Snippet: Glutathione S -transferase-fused PGK1 protein was coincubated with HA-tagged KAT2B protein purified from HEK293T cells in reaction buffer (50 mM HEPES, pH 7.8, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM dithiothreitol), supplemented with 20 mM lactyl-CoA (HY-141540, MCE, USA) [ ].

Techniques: Injection, Control, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Western Blot

Journal: bioRxiv

Article Title: Functionally Mature Bioengineered Human Skeletal Muscle Tissues Capture Essential Aspects of Glucose Metabolism

doi: 10.64898/2026.01.16.698404

Figure Lengend Snippet: (A) Effect of acute treatment with clenbuterol (10 nM, 20 min) on phosphorylation of β-adrenergic (B2AR) downstream targets including HSL (Ser660), CREB (Ser133), and RPS6 (Ser235/236) (n=4). (B) Gene expression of B2AR–responsive transcripts ( PPARGC1A , NR4A3 , NR4A1 , PGK1 , GYS1 , and others) after chronic clenbuterol treatment at the indicated doses (n=5). (C) Maximal tetanic force measured over time in tissues exposed to increasing clenbuterol concentrations (0.2–10 nM). Right: day-7 maximal force relative to vehicle for each dose (n=3). (D) Representative confocal images of myofibrillar structure in vehicle- and clenbuterol-treated tissues (n=2) (E) Maximal tetanic force during an inactivity protocol with or without clenbuterol treatment. Left: force traces over the detraining period. Right: day-7 maximal force in vehicle versus clenbuterol-treated groups (n=5). (F) Fatigue development during repeated contractions (protocol 2) in glucose-containing media in control versus clenbuterol-treated tissues. Inset: endurance values at the end of the protocol (n=5). (G) Fatigue development during repeated contractions (protocol 2) in glucose-free media in control versus clenbuterol-treated tissues (n=5). Data are presented as mean ± SEM, with individual values shown where applicable. Statistical comparisons were performed using one-way or two-way ANOVA with appropriate multiple-comparisons correction. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following primers were used: TaqMan Gene Expression Assays (ThermoFisher Cat# 4331182), with the following ID numbers: GYS1: ID Hs00157863_m1, Ppargc1a: ID Hs00173304_m1, Nr4a3: ID Hs00545009_g1, Nr4a1: ID Hs00374226_m1 and Pgk1: ID Hs00943178_g1.

Techniques: Phospho-proteomics, Gene Expression, Control

Journal: Oncogenesis

Article Title: PRAS40 promotes colorectal cancer stemness by enhancing glycolysis through triggering PGK1 acetylation

doi: 10.1038/s41389-025-00594-x

Figure Lengend Snippet: A Co-IP followed by western blotting analyses in PRAS40-overexpressed HT29 cells. Analyses of PGK1 activity in HT29 cells with PRAS40 knockdown B or overexpression C . D – H HT29 cells were introduced with empty vector or FLAG-PRAS40 expression vector together with control or PGK1 shRNA. Glucose consumption analyses D , lactate production analyses E , flow cytometry analyses with anti-CD44 and anti-CD133 antibodies F were conducted. Representative images and quantification of tumor spheres were shown G , H . Data represent the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies were purchased for detection of PRAS40 (2691S), PCNA (13110), PCAF (3378 T) (Cell Signaling); PGK1 (17811-1-AP), CD133 (66666-1-Ig), α-Tubulin (66031-1-Ig), β-Actin (66009-1-Ig), CD44 (15675-1-AP) (Proteintech); SIRT7 (ab259968), anti-phosphotryosine (ab179530) (Abcam); and pan-Acetyl (sc-8649)(Santa Crus Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Activity Assay, Knockdown, Over Expression, Plasmid Preparation, Expressing, Control, shRNA, Flow Cytometry

Journal: Oncogenesis

Article Title: PRAS40 promotes colorectal cancer stemness by enhancing glycolysis through triggering PGK1 acetylation

doi: 10.1038/s41389-025-00594-x

Figure Lengend Snippet: Co-IP followed by western blotting analyses in HT29 cells with PRAS40 knockdown A or overexpression B . C – I HT29 cells were introduced with empty vector or FLAG-PRAS40 expression vector, and treated with or without acetyltransferase inhibitors PU139 (20 μM) or Anacardic acid (5 or 10 μM). NC, negative control. Analyses of PGK1 activity C , representative images and quantification of tumor spheres were shown D , E , real time PCR analyses F , western blotting analyses G , and flow cytometry analyses with anti-CD44 and anti-CD133 antibodies H , I were performed. J Co-IP followed by western blotting analyses in HT29 cells. Co-IP followed by western blotting analyses in HT29 cells with PRAS40 knockdown K or overexpression L . Data represent the mean ± SD. ** P < 0.01; *** P < 0.001. The quantification results of the band density were labeled above the bands.

Article Snippet: Antibodies were purchased for detection of PRAS40 (2691S), PCNA (13110), PCAF (3378 T) (Cell Signaling); PGK1 (17811-1-AP), CD133 (66666-1-Ig), α-Tubulin (66031-1-Ig), β-Actin (66009-1-Ig), CD44 (15675-1-AP) (Proteintech); SIRT7 (ab259968), anti-phosphotryosine (ab179530) (Abcam); and pan-Acetyl (sc-8649)(Santa Crus Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knockdown, Over Expression, Plasmid Preparation, Expressing, Negative Control, Activity Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Labeling