Journal: Oncogenesis

Article Title: BRG1 inhibits glycolysis by promoting SHP1-mediated dephosphorylation of PKM2 in non-small cell lung cancer

doi: 10.1038/s41389-025-00577-y

Figure Lengend Snippet: A HEK293T cells were transfected with Flag-PKM2 plasmid, and then transfected with vector, HA-PTPN2, HA-SHP1 or HA-SHP2 plasmids. After EGF stimulation, whole cell lysates (WCL) were collected and subjected to immunoprecipitation using anti-Flag magnetic beads, followed by immunoblot analysis. B , C The protein level of p-PKM2 Y105 was detected by western blot assays in control or SHP1, SHP2 overexpression A549 cells with or without EGF stimulation. D , E Co-IP assays were performed in A549 and PC9 cells using anti-BRG1 or anti-SHP1 antibodies to examine the endogenous interaction between BRG1 and SHP1. F Co-IP assays were performed in HEK293T cells using antibodies against GST or HA to examine the exogenous interaction between GST-BRG1 and HA-SHP1. G HEK293T cells were transfected with Flag-PKM2 plasmid, along with HA-SHP1, with or without various amounts of GST-BRG1 (2, 4, and 6 μg). Whole cell lysates (WCL) were collected and subjected to immunoprecipitation using anti-Flag magnetic beads, followed by immunoblot analysis. The interaction between Flag-PKM2 and HA-SHP1 in control or EGF-stimulated cells was analyzed. H HEK293T cells were transfected with or without various amounts of GST-BRG1 (2, 4, and 6 μg). Whole cell lysates (WCL) were collected and subjected to immunoprecipitation using Protein A/G Magnetic Beads with anti-PKM2 antibodies, followed by immunoblot analysis. The interaction between PKM2 and SHP1 in control or EGF-stimulated cells was analyzed. I The protein level of p-PKM2 Y105 was detected in control (shNC) or shSHP1-treated A549 cells with or without BRG1 overexpression under EGF stimulation by western blot assays. All western blotting experiments were independently replicated three times, yielding consistent results.

Article Snippet: BRG1(3508; Cell Signaling Technology), PKM2(3198; Cell Signaling Technology), p-PKM2 Y105 (3827; Cell Signaling Technology), Flag tag(14793S; Cell Signaling Technology), GST tag(AE001;Abclonal), HA tag (AE008; Abclonal), β-actin (AC038; Abclonal), LAMIN B1(A16909; Abclonal), SHP1(A19111;Abclonal), SHP2(A19112; Abclonal), TEPP-46(HY-18657; MedChemExpress), Anti-GST Magnetic Beads (HY-K0222; MedChemExpress), Anti-Flag Magnetic Beads(HY-K0207; MedChemExpress), Anti-HA Magnetic Beads (HY-K0201; MedChemExpress), Protein A/G Magnetic Beads(HY-K0202; MedChemExpress) were commercially purchased.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Over Expression, Co-Immunoprecipitation Assay

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Hexokinase II and PKM2 protein expression in human normal skin and malignant melanoma tissues, and relative protein expression. ( A ) Western blot analysis. Levels of HK II and PKM2 were markedly higher in melanoma tissues than in normal tissues. β-actin served as a loading control. ( B ) Quantitative comparison of relative HK II and PKM2 protein levels in normal ( n = 6) and melanoma tissues ( n = 6). Statistical significance was determined using Tukey’s post hoc multiple comparison tests (**** p < 0.0001). HK II: Hexokinase II, and pyruvate kinase M2: PKM2.

Article Snippet: Primary antibodies against Cyclin D1 (92G2, #2978), Cyclin E1 (D7T3U, #20808), CDK4 (D9G3E, #12790), HK II (C64G5, #2867), PKM2 (D78A4, #4053), Bax (#5023), Bcl-2 (#2820), Cleaved Caspase-3 (Asp175, #9664), Caspase-3 (D3R6Y, #14220), Phospho-RIP (Ser166, D1L3S, #65746), RIP (D94C12, #3493), Phospho-MLKL (Ser358, D6H3V, #91,689), and MLKL (D2I6N, #14,993) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Resveratrol modulates glycolytic enzymes and induces apoptosis and necroptosis in malignant melanoma cells. G361 and SK-MEL-24 cells were treated with RSV (0, 20, 40, 60 µM) for 48 h. ( A ) Western blot analysis of HK II, PKM2, and apoptosis-related proteins (cleaved caspase-3, caspase-3, Bax, and Bcl-2). β-Actin served as loading control. ( B ) Western blot analysis of necroptosis-associated proteins, including phosphorylated MLKL (p-MLKL) and phosphorylated RIP (p-RIP). Total MLKL and total RIP were used as loading controls. ( C ) Intracellular ATP levels. ( D ) Hexokinase enzymatic activity. ( E ) Caspase-3/7 activity. Data are expressed as mean ± SE ( n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). HK II: Hexokinase II; PKM2: pyruvate kinase M2; RSV: resveratrol.

Article Snippet: Primary antibodies against Cyclin D1 (92G2, #2978), Cyclin E1 (D7T3U, #20808), CDK4 (D9G3E, #12790), HK II (C64G5, #2867), PKM2 (D78A4, #4053), Bax (#5023), Bcl-2 (#2820), Cleaved Caspase-3 (Asp175, #9664), Caspase-3 (D3R6Y, #14220), Phospho-RIP (Ser166, D1L3S, #65746), RIP (D94C12, #3493), Phospho-MLKL (Ser358, D6H3V, #91,689), and MLKL (D2I6N, #14,993) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Control, Activity Assay

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Proposed mechanism of resveratrol-induced cell death in malignant melanoma cells. Resveratrol decreases HK II and PKM2 expression, resulting in reduced glycolysis-dependent ATP production. The subsequent decline in cellular energy availability induces mitochondrial dysfunction and promotes intrinsic apoptosis through an increased Bax/Bcl-2 ratio and activation of caspase-3/7. Simultaneously, metabolic stress triggers necroptosis through phosphorylation of RIP, and MLKL. These coordinated mechanisms collectively contribute to cell death in melanoma cells.

Article Snippet: Primary antibodies against Cyclin D1 (92G2, #2978), Cyclin E1 (D7T3U, #20808), CDK4 (D9G3E, #12790), HK II (C64G5, #2867), PKM2 (D78A4, #4053), Bax (#5023), Bcl-2 (#2820), Cleaved Caspase-3 (Asp175, #9664), Caspase-3 (D3R6Y, #14220), Phospho-RIP (Ser166, D1L3S, #65746), RIP (D94C12, #3493), Phospho-MLKL (Ser358, D6H3V, #91,689), and MLKL (D2I6N, #14,993) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay, Phospho-proteomics

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 4. HA344-induced cell death in CMM is initiated by PKM covalent targeting. A, Chemical structures of VG41 and VG43. B, Cell viability measured by trypan blue exclusion of A375S, A375R#1, A375R#2, SKMEL28, and SKMEL28R CMM treated with HA344, VG41, or VG43 at 2 mmol/L for 48 hours. C, Number of peptides according to peptide intensity of A375S cells click chemistry treated 1 hour with VG41 at 2 mmol/L. D, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of A375S and A375R#1 cell click chemistry. E, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of SKMEL28 and SKMEL28R cell click chemistry. F, Table of mass spectrometry analysis of recombinant PKM2 treated or not with HA344 (2 mmol/L, 1 hour). G, PKM2 and GAPDH Western blot analysis of A375S cell CETSA and their quantification. H, PKM2 and GAPDH Western blot analysis of A375R#1 cell CETSA and their quantification. , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Western Blot, Mass Spectrometry, Recombinant

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 5. HA344 inhibits glycolytic and nonglycolytic functions of PKM2 and IMPDH activity in CMM. A, PK enzymatic activity under HA344 (at 0.5, 1, and 2 mmol/L) and Shikonin (at 2 mmol/L) treatment. B, PK enzymatic activity of HEK293T cells of EV, PKM2, or PKM2 C424 L after GFP trap treated or not with HA344. C, PKM2 monomer and dimer Western blot analysis of A375S and A375R#1 cells treated 30 minutes, 3 hours, or 6 hours with HA344 at 2 mmol/L. D, Immunofluorescence PKM2 staining of A375S treated 30 minutes, 1 hour, and 6 hours with HA344 at 2 mmol/L, measured by confocal imaging. E, Cell-cycle analysis of A375R#1 treated 6 hours with HA344 at 2 mmol/L (top) and cyclin D1 Western blot analysis of A375S and A375R#1 (bottom). F, Glycolysis relative to control metabolite levels of A375S (top) and A375R#1 (bottom) treated 3 or 6 hours with HA344 at 2 mmol/L and 3 hours with Shikonin at 2 mmol/L. G, Unlabeled (Mþ0) and 13C-labeled glucose 6-P (Mþ6; left) and pyruvate (Mþ3; right) of A375S cells treated or not with HA344 at 2 mmol/L 3 hours and 6 hours and Shikonin 2 mmol/L. H, Unlabeled (Mþ0) and 13C-labeled glucose 6-P (Mþ6; left) and pyruvate (Mþ3; right) of A375R#1 cells treated or not with HA344 at 2 mmol/L 3 hours and 6 hours and Shikonin 2 mmol/L. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Activity Assay, Western Blot, Staining, Imaging, Cell Cycle Assay, Control, Labeling

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 6. HA344 reduces tumor growth in mice xenografted with CMM. A, A375S tumor growth in control and HA344-treated mice at 5 mg/kg/day. Tumor size is presented as the mean SEM, with n ¼ 6 mice in each group. Statistical significance was assessed by a two-way ANOVA with a Sidak multiple comparisons test. B, A375R#2 tumor growth in control and HA344-treated mice at 5 mg/kg/day). Tumor size is presented as the mean SEM with n ¼ 6 mice in each group. Statistical significance was assessed by a two-way ANOVA with a Sidak multiple comparisons test. C, Frozen sections of A375S tumors from mice treated with vehicle or HA344 were fixed and stained using PKM2 antibody (Alexa 594) and DAPI (blue). The experiment was repeated on sections from all mice per condition. Quantifications of PKM2 fluorescence intensity are presented in a.u. and represent the mean/field SEM of at least four independent fields. Statistical significance was assessed by an unpaired t test. D, Frozen sections of A375R#2 tumors from mice treated with vehicle or HA344 were fixed and stained using PKM2 antibody (Alexa 594) and DAPI (blue). The experiment was repeated on sections from all mice per condition. Quantifications of PKM2 fluorescence intensity are presented in a.u. and represent the mean/field SEM of at least four independent fields. Statistical significance was assessed by an unpaired t test. E, Quantification of PKM2 Western blot analysis of A375S xenografts in CT NaCl and HA344-treated tumors. F, Quantification of PKM2 Western blot analysis of A375R#2 xenografts in CT NaCl and HA344-treated tumors. G, Three mg of A375S tumor proteins from control (n ¼ 6) and HA344 (n ¼ 5)-treated groups were used to measure PKM activity (n ¼ 3 measures for each tumor). PKM activity is presented in RFU and as the mean SEM of tumors. H, Three mg of A375R#2 tumor proteins from control (n ¼ 6) and HA344 (n ¼ 5)-treated groups were used to measure PKM activity (n ¼ 3 measures for each tumor). PKM activity is presented in RFU and as the mean SEM of tumors. , P < 0.05; , P < 0.001; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Control, Staining, Western Blot, Activity Assay

Journal: Cancer Research

Article Title: Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma

doi: 10.1158/0008-5472.can-20-2114

Figure Lengend Snippet: Figure 7. HA344 induces cell death and targets PKM in patients with CMM. A, The SKCM TCGA dataset from Oncolnc was analyzed for PKM mRNA expression and OS. Patients were stratified according toPKM expression and BRAFV600E mutation. B, TheGSE3189 TCGA dataset from the GEO database was analyzed for PKM mRNA expression according to melanocyte transformation status. Statistical significance was determined using an unpaired t test. C, DAPI staining of CMM patients’short-term cultures treated 48 hours with vemurafenib (Vemu). D, DAPI staining of CMM patients’ short-term cultures treated 48 hours with HA344. E, Cell viability measured by trypan blue exclusion of patient#1, patient#6S, and patient#6R treated with HA344, VG43, and VG41. F, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of patient#1 click chemistry. G, Western blot analysis of PKM2, PKM1, IMPDH2, and GAPDH, of patient#6S and patient# 6R click chemistry. H, Confocal images of PKM2 staining of patient#1 cells treated 30 minutes, 1 hour, and 6 hours with HA344 at 2 mmol/L. , P < 0.05; , P < 0.01; , P < 0.0001.

Article Snippet: Mutation of PKM2 and transfection in HEK293 cells The PKM2 vector pEGFP-C1-PKM2WT was purchased from Addgene (Plasmid #64698).

Techniques: Expressing, Mutagenesis, Transformation Assay, Staining, Western Blot

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Representative immunofluorescence images and quantification of glycolytic enzymes PKM2 and LDHA in human PLL and OPLL tissues. Scale bar = 100 µm, n = 3. B Western blot analysis of GLUT1, PKM2, and LDHA protein levels in PLL and OPLL cells. n = 3. C , D . Glucose uptake and lactate production in PLL and OPLL cells. n = 3. E ECAR measured by Seahorse XF Analyzer to assess the glycolytic capacity of PLL and OPLL cells. n = 3. F Representative images showing TMRE staining (orange-red) in PLL and OPLL cells. The bar graph depicts the quantification of relative TMRE fluorescence intensity. Scale bar = 30 µm, n = 3. G Western blot analysis of PDK1, Cyto c, and ATP5A in OPLL and PLL cells. H Intracellular ROS levels measured by DCFH-DA fluorescence. Scale bar = 50 µm. I OCR measured by Seahorse XF Analyzer to assess mitochondrial respiration. n = 3. J Western blot analysis of RUNX2, OSX, and ALP during a 15day time course of osteogenic differentiation of ligament cells. K Western blot analysis of GLUT1, HK2, and LDHA during a 15day time course of osteogenic differentiation of ligament cells. L Western blot analysis of PDK1, Cyto c, and ATP5A during a 15day time course of osteogenic differentiation of ligament cells. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Immunofluorescence, Western Blot, Staining, Fluorescence, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Venn diagram illustrating the overlap of hub genes identified from the PPI network using five different centrality algorithms: MCC, DMNC, EPC, MNC, and Degree. B Key genes identified by the DMNC method within the PPI network. C Correlation analysis between the glycolysis flux score and ADAM12 expression in OPLL and PLL tissues. D Distribution of ADAM12 across the major cell types in posterior longitudinal ligament tissue. E Scatter plot showing the positive correlation between ADAM12 expression and the glucose metabolism pathway activity score across individual ligament cells. F Representative immunofluorescence images and quantification of ADAM12 expression in human OPLL and PLL tissues. Scale bar = 50 μm, n = 3. G Representative immunofluorescence images showing co-localization of ADAM12 (green) and PKM2 (red) in OPLL tissue. Scale bar = 30 μm. H , I Quantitative PCR analysis of ADAM12L and ADAM12S mRNA levels in PLL and OPLL cells. n = 3. J Quantitative PCR analysis of the expression levels of the ADAM12L and ADAM12S isoforms in OPLL cells. n = 3. K Western blot analysis of ADAM12 protein levels in PLL and OPLL cells. n = 3. L ELISA analysis of ADAM12 protein levels in PLL and OPLL ligament cells supernatant. n = 5. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Expressing, Activity Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Heatmap showing the relative abundance of central carbon metabolites in control (NC), osteogenically differentiated (NC-OD), and ADAM12-overexpressing osteogenically differentiated (OE-OD) ligament cells. n = 3. B Box plots quantifying the levels of key glycolytic intermediates (fructose-1,6-bisphosphate, 3-phosphoglycerate, pyruvate, and lactate) from the metabolomics data. n = 3. C , D Western blot analysis of glycolytic markers (GLUT1, HK2, PKM2, LDHA) and mitochondrial markers (PGC1α, mtTFA, ATP5A, cytochrome c) in ligament cells after ADAM12 knockdown and overexpression. n = 3. E , F Glucose uptake capacity in ligament cells after ADAM12 knockdown and overexpression. n = 3. G , H Lactate production in ligament cells after ADAM12 knockdown and overexpression. n = 3. I , J Intracellular pyruvate content in ligament cells after ADAM12 knockdown and overexpression. n = 3. K , L ECAR measured by Seahorse XF Analyzer in ligament cells after ADAM12 knockdown and overexpression. n = 3. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Control, Western Blot, Knockdown, Over Expression, Standard Deviation

Journal: Biomaterials

Article Title: Dual inhibition of oxidative phosphorylation and glycolysis using a hyaluronic acid nanoparticle NOX inhibitor enhanced response to radiotherapy in colorectal cancer.

doi: 10.1016/j.biomaterials.2025.123437

Figure Lengend Snippet: Fig. 3. Combination of HANP/GKT831 inhibited tumor progression and impacted metabolism with radiotherapy. HANP/GKT831 enhanced sensitivity to radiation and suppressed cell migration and invasion in the MC38 mouse tumor cells. A. Colony formation assay detected cell colonies resistant to 2 Gy of RT (n = 3). B. Transwell migration assay evaluated cell migration following the treatments. Cells were treated with 0.01 μM of conventional GKT831 or HANP/GKT831 con taining 0.01 μM of equivalent doses of GKT831. Quantification of 2D migration assay after 48 h in culture (n = 5). HANP/GKT831 reduced the protein levels of representative signal molecules in glycolysis, mitochondrial OXPHOS, and DNA repair pathways. C. The levels of Western blot analysis of NOX1, NOX4, glycolysis related (Hexokinase 2 and PKM2), and mitochondrial OXPHOS associated proteins (MT-ATP6 and MT-ND1). D. The protein levels in the cell cycle and DNA repair pathway, Cyclin D1, MSH6, and CHK1 proteins, were examined from the treated tumor lysates by Western blot analysis. β-actin served as the loading control. Similar results were obtained from at least 3 repeated studies. Student’s t-test: *p < 0.05 and ****p < 0.0001.

Article Snippet: After transferring to a polyvinylidene difluoride and blocking in 5 % of milk in tris-buffered saline (TBS) for 1 h, the blots were probed with rabbit anti-NOX1 antibody (MBS9609001, MYBioSource, San Diego, CA, USA), rabbit anti-NOX4 antibody (MBS820230, MYBioSource), anti-Hexokinase II rabbit monoclonal antibody (C64G5, Cell Signaling Technology), anti PKM2 XP rabbit monoclonal antibody (D78A4, Cell Signaling Technology), rabbit anti-MT-ATP6 antibody (70262, Cell Signaling Technology), rabbit anti-MT-ND1 antibody (NBP2-94462, NOVUS, MO, USA), anti-Cyclin D1 rabbit monoclonal antibody (2978, Cell Signaling Technology), rabbit anti-MSH6 antibody (MBS9626584, MYBioSource), rabbit anti-Phospho-Chk1 (Ser345) antibody (#2341, Cell Signaling Technology), hamster anti MCP-1 (CCL2) antibody (505911, Biolegend, San Diego, CA, USA), rabbit antiCXCL10 monoclonal antibody (701225, Invitrogen), anti-β-Actin mouse monoclonal antibody (A5316, Sigma-Aldrich).

Techniques: Migration, Colony Assay, Transwell Migration Assay, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Representative images comparing whole kidney size between Glis3-KO2 kidneys treated with vehicle or compound 3K are shown. b , Violin plot depicting the KW/BW ratio (%) for WT and Glis3 -KO2 mice treated with vehicle or compound 3K. n ≥ 10; **** P < 0.0001. c , Representative hematoxylin and eosin-scanned images of kidney sections from Glis3 -KO2 kidneys treated with vehicle or compound 3K. Bars indicate 1 mm. d , Comparison of cystic index between Glis3 -KO2 kidneys treated with vehicle or compound 3K. Cystic index represents the percentage of renal tissue occupied by cysts. Data are presented as mean ± s.e.m., n = 6 . ** P < 0.01. e , Comparison of renal cyst size (mm 2 ) between kidneys from Glis3 -KO2 mice treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n = 6; * P < 0.05. f , Comparison of the number of cysts per kidney section between Glis3 -KO2 kidneys treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n = 8; ** P < 0.01. g , Comparison of serum creatinine levels (mg/dl) between WT and Glis3 -KO2 mice treated with vehicle or compound 3K. h , RT–qPCR analysis of Pkm2 , c-Myc , Hk2 , Havcr1 and Lcn2 between WT and Glis3 -KO2 mice treated with vehicle or compound 3K. Data are presented as mean ± s.e.m., n ≥ 5; **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. i , Schematic illustration depicting the relationship between loss of GLIS3 function, regulation of PKM2 and cystogenesis. GLIS3 deficiency enhances Pkm gene expression in kidneys with a preferential increase in the Pkm2 isoform. Increased PKM2 phosphorylation at S37 and Y105 promotes dimer formation. PKM2-S37 phosphorylation is facilitated by increased pERK1/2 levels. Together, these events promote glycolysis, cell proliferation and cystogenesis in GLIS3-deficient kidneys. Figure 6i was created using BioRender.com.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Comparison, Quantitative RT-PCR, Gene Expression, Phospho-proteomics

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , b , Analysis of RNA-seq transcripts per million (TPM) values for PKM2 transcripts ( a ) and PKM2:PKM1 transcript ratio ( b ) in PND7, 14 and 28 WT and Glis3 -KO2 kidneys. c , Sashimi analysis plot showing the alternatively spliced isoforms of PKM2 and PKM1. Exons (darker color) and splice junctions (lighter color) from WT are in blue and those from Glis3 -KO2 are in purple. Splice junction read counts are numbered above their respective ribbons. STAR genomic alignment counts scaled to 300 M mappable reads per sample group labels the y axis, exon and splice junction coordinates are on the x axis and mRNA isoforms are shown on the bottom (exons in black and introns as lines). d , Analysis of the PKM2-specific splice junction read counts, plotted for visualization between PND7, 14 and 28 WT and Glis3 -KO2 kidneys. e , f , Exogenous expression of GLIS3 in WT and Glis3 -KO2 RECs decreased Pkm2 mRNA expression ( e ) but did not change Pkm1 mRNA expression ( f ). RECs were infected with Glis3 lentivirus for 36 h, and gene expression was analyzed by RT–qPCR. Data are presented as mean ± s.e.m., n ≥ 5 ; **** P < 0.0001, *** P < 0.001; ** P < 0.01; * P < 0.05. n.s., nonsignificant. g , RT–qPCR for Pkm2 from isolated collecting duct (CD) and proximal tubule (PT) cells. Data are presented as mean ± s.e.m., n ≥ 3; * P < 0.05.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: RNA Sequencing, Expressing, Infection, Gene Expression, Quantitative RT-PCR, Isolation

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Immunoblot analysis of total PKM2 (GA−) and glutaraldehyde (GA+) crosslinked PKM2 expression. The relative level of PKM2 dimers were quantified by densitometric analysis. b , Immunoblot analysis of PKM2(pY105) and total PKM2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 3 ; ** P < 0.01; * P < 0.05. c , Immunoblot analysis of PKM2(pS37) and total PKM2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 3 ; ** P < 0.01; * P < 0.05. d , Representative images of PND28 WT and Glis3 -KO2 kidney sections immunostained for PKM2(pS37) showing enhanced nuclear staining in renal cysts. The inlets denote the location of zoomed-in images. DBA marks collecting ducts and LTL marks proximal tubules. e , Immunoblot analysis of pERK1/2 and total ERK1/2 protein expression in WT and Glis3 -KO2 kidneys. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 4; ** P < 0.01.

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Western Blot, Expressing, Staining

Journal: Experimental & Molecular Medicine

Article Title: Regulation of PKM2 expression and function by GLIS3 during metabolic reprogramming in polycystic kidneys

doi: 10.1038/s12276-026-01676-5

Figure Lengend Snippet: a , Immunoblot analysis of PKM2 protein levels in WT and Glis3 -KO2 RECS 3 days after siRNA-mediated PKM2-KD. Protein expression was quantified by densitometric analysis. Data are presented as mean ± s.e.m., n = 5; *** P < 0.001; ** P < 0.01. b , Analysis of lactate production in media from primary WT and Glis3 -KO2 RECs with or without PKM2-KD. c , Glycolytic rate was measured in primary WT and Glis3 -KO2 REC mice with or without PKM2-KD using a Seahorse analyzer after sequential injections of rotenone/antimycin A and 2-DG. d , e , Basal ( d ) and compensatory ( e ) glycolysis were calculated and plotted ( n = 3). f , Representative images of WT and Glis3 -KO2 REC spheroids with and without PKM2-KD at 5 and 9 days. Bars indicate 50 μm. g , Violin plot showing the size (µm) distribution of the spheroids generated at day 5 from WT and Glis3 -KO2 RECs with or without PKM2-KD ( n ≥ 4). Each data point represents an individual spheroid measurement. * P < 0.05, ** P < 0.01, *** P < 0.001. h , Spheroid images at day 5 were taken using the EVOS M7000, and spheroid diameter and number were analyzed using ImageJ and plotted according to size distribution—either 30–50, 50–100 or >100 μm. Total indicates the number of spheroids analyzed in each group ( n = 39–164). i , Violin plot showing the size (µm) distribution of the spheroids generated at day 9 from WT and Glis3 -KO2 REC mice with or without PKM2-KD ( n ≥ 4). Each data point represents an individual spheroid measurement. * P < 0.05, ** P < 0.01, *** P < 0.001. j , Day 10 spheroid size distribution—either 30–50, 50–100 or >100 μm. Total indicates the number of spheroids analyzed in each group ( n = 60–191). k , Representative image of the size of WT and Glis3 -KO2 REC spheroids 5 days following treatment with vehicle (0.1% DMSO) or compound 3K (1 μM). Bars indicate 50 μm. l , Violin plot showing the size (µm) distribution of the spheroids generated from the RECs of WT and Glis3 -KO2 kidneys ( n ≥ 4). Each data point represents an individual spheroid measurement. **** P < 0.0001. m , Size distribution—30–50, 50–100 or >100 μm of WT and Glis3 -KO2 REC spheroids with or without PKM2 inhibition. Total indicates the number of spheroids analyzed in each group (n = 39–164).

Article Snippet: To examine the effect of PKM2 inhibition on cyst formation, PND7 Glis3 -Pax8Cre mice were treated intraperitoneally with the PKM2 inhibitor, compound 3K (S8616; Selleckchem Chemicals) (10 mg/kg) or vehicle control (10% dimethylsulfoxide (DMSO), 90% corn oil) every 24 h for 7 days.

Techniques: Western Blot, Expressing, Generated, Inhibition