Journal: PLOS ONE

Article Title: USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

doi: 10.1371/journal.pone.0290688

Figure Lengend Snippet: (A) Top: 293T cells were transfected with Flag-USP4 and Myc-PKM2, immunoprecipitated with Myc antibody, and immunoblotted with Flag antibody. Bottom: 293T cells were transfected with Flag-USP4 and Myc-PKM2, immunoprecipitated with Flag antibody, and immunoblotted with Myc antibody. (B) Endogenous PKM2 was immunoprecipitated from either AGS cells (top) or HGC-27 cells (bottom) with the USP4 antibody and immunoblotted with the PKM2 antibody. (C) Top: Schematic representation of flag-tagged full-length USP4 (FL) and its various deletion mutants (M1–M8). Middle: The expression of flag-tagged full-length USP4 (FL) and its various deletion mutants (Input). Bottom: Flag-USP4 and its eight deletion mutants were co-transfected into 293T cells with Myc-tagged PKM2, respectively, immunoprecipitated with Myc antibody, and immunoblotted with Flag antibody. (D) Top: Schematic representation of Myc-tagged full-length PKM2 (FL) and its five deletion mutants. Middle: The expression of Myc-tagged PKM2 and its five deletion mutants (input). Bottom: Myc-PKM2 and its five deletion mutants were co-transfected into 293T cells with Flag-tagged USP4, respectively, immunoprecipitated with Myc antibody, and immunoblotted with Flag antibody. (E) Immunofluorescent staining of USP4 (green) and PKM2 (red) in 293T cells transfected with Flag-USP4 and Myc-PKM2. The left panel is nuclear 4’,6-diamidino-2-phenylindole (DAPI; blue). The right panel is the overlay of USP4 (green), PKM2 (red), and DAPI (blue) staining of the same field. Scale bar: 10 m.

Article Snippet: USP4 shRNAs and PKM2 shRNAs were purchased from Genecopoeia.

Techniques: Transfection, Immunoprecipitation, Expressing, Staining

Journal: PLOS ONE

Article Title: USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

doi: 10.1371/journal.pone.0290688

Figure Lengend Snippet: (A) Immunoblotting of USP4 (Flag) and PKM2 in AGC cells that are stably transfected with control plasmids, Flag-USP4 and Flag-USP4 mutant, respectively. (B) Immunoblotting of USP4 (Flag) and PKM2 in HGC-27 cells that are stably transfected with control plasmid and Flag USP4, respectively. (C) Immunoblotting of USP4 and PKM2 in AGS cells that are stably transfected with scramble plasmid, USP4 shRNA (clone 1), and USP4 shRNA (clone 3), respectively. (D) Immunoblotting of USP4 and PKM2 in HGC-27 cells that are stably transfected with scramble plasmids, USP4 shRNA (clone 1) and USP4 shRNA (clone 3), respectively. (E) 293T cells were co-transfected with Myc-PKM2, HA-ubiquitin (Ub), and Flag-USP4 or the USP4 C311A mutant, immunoprecipitated with Myc beads, and immunoblotted with antibodies to HA and Myc. Cells were treated with MG132 (10 M) for 4 hours before harvest.

Article Snippet: USP4 shRNAs and PKM2 shRNAs were purchased from Genecopoeia.

Techniques: Western Blot, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, shRNA, Immunoprecipitation

Journal: PLOS ONE

Article Title: USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

doi: 10.1371/journal.pone.0290688

Figure Lengend Snippet: (A) Left: The cell proliferation of USP4- or USP4 C331A -transfected 293T cells Middle: The cell proliferation of AGS cells transfected with USP4. Right: The cell proliferation of HGC-27 cells transfected with USP4. (B) The cell proliferation of AGS cells transfected with USP4 shRNAs. (C) The cell proliferation of AGS cells transfected with USP4 and PKM2 shRNAs. (D) Representative images and statistical results of colony formation in AGS cells transfected with USP4. (E) Representative images and statistical results of colony formation in AGS cells transfected with USP4 shRNAs. (F) Representative images and statistical results of colony formation in AGS cells transfected with USP4 and PKM2 shRNAs.

Article Snippet: USP4 shRNAs and PKM2 shRNAs were purchased from Genecopoeia.

Techniques: Transfection

Journal: PLOS ONE

Article Title: USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

doi: 10.1371/journal.pone.0290688

Figure Lengend Snippet: (A) The expression level of Glut1 and LDHA in USP4 overexpression and USP4 knockdown cells; (B) the glucose uptake of AGS cells when USP4 is overexpressed or knocked down; (C) the lactate production of AGS cells when USP4 is overexpressed or knocked down; (D) the glucose uptake and lactate production in AGS cells in which USP4 is overexpressed but PKM2 is knocked down.

Article Snippet: USP4 shRNAs and PKM2 shRNAs were purchased from Genecopoeia.

Techniques: Expressing, Over Expression

Journal: PLOS ONE

Article Title: USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

doi: 10.1371/journal.pone.0290688

Figure Lengend Snippet: USP4 increases PKM2 stability via deubiquitination. Ub = ubiquitin.

Article Snippet: USP4 shRNAs and PKM2 shRNAs were purchased from Genecopoeia.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: Knockdown of PKM2 inhibits osteoclast differentiation of BMMs. BMMs were infected with an adenovirus carrying PKM2-specific shRNA or a control adenovirus to verify the expression of PKM2 mRNA ( A ) and protein ( B ) in the cells. C , relative quantification of PKM2 protein. Knockdown of PKM2 inhibited osteoclast differentiation ( D ) and bone resorption function ( F ) of BMMs. E , number of TRAP-positive cells. G , quantification of bone resorption area. Cells were intervened and cultured for 7 days, cell morphology was observed ( H ), and number of actin rings is counted ( I ). Data are expressed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. BMM, bone marrow–derived macrophage; PKM2, pyruvate kinase M2; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Knockdown, Infection, shRNA, Control, Expressing, Quantitative Proteomics, Cell Culture, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: Knockdown of PKM2 inhibits the expression of osteoclast-related genes and proteins. The expression of mRNA ( A ) and protein ( B ) of osteoclast-related genes, CTSK, NFATc1, and TRAP, in BMMs was inhibited after the knockdown of PKM2. C , relative quantification of CTSK, NFATc1 and TRAP protein. Data are displayed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. BMM, bone marrow–derived macrophage; CTSK, cathepsin K; NFATc1, nuclear factor of activated T cell 1; PKM2, pyruvate kinase M2; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Knockdown, Expressing, Quantitative Proteomics, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: PKM2 activators, DASA-58 and TEPP-46, inhibit osteoclast differentiation of BMMs. BMMs were treated with different concentrations of PKM2 activators DASA-58 ( A ) and TEPP-46 ( B ), respectively, and MTT assays showed their effect on cell proliferation. The TRAP staining showed that osteoclast differentiation of BMMs was inhibited by treatment with appropriate concentrations of PKM2 activators DASA-58 ( C ) and TEPP-46 ( E ), respectively. Number of TRAP-positive cells by treatment with appropriate concentrations of DASA-58 ( D ) and TEPP-46 ( F ). G , the representative TRAP staining images of BMMs after treating with PKM2 activators at different time points. Number of TRAP-positive cells after treating with DASA-58 ( H ) and TEPP-46 ( I ) at different time points. Data are expressed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. BMM, bone marrow–derived macrophage; PKM2, pyruvate kinase M2; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Staining, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: PKM2 activators, DASA-58 and TEPP-46, inhibit osteoclast-related gene expression. A and B , osteoclast differentiation was inhibited when cells were treated with PKM2 activator DASA-58. A , cells were intervened and cultured for 7 days, and cell morphology was observed. B , number of actin rings by treatment with DASA-58 (10 μM). The expression of osteoclast-associated genes, CTSK, NFATc1, TRAP, and MMP-9, was inhibited after the addition of PKM2 activators DASA-58 ( C ) and TEPP-46 ( D ). DASA-58 and TEPP-46 were used at 10 μM in the culture medium. Data are displayed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. BMM, bone marrow–derived macrophage; CTSK, cathepsin K; NFATc1, nuclear factor of activated T cell 1; PKM2, pyruvate kinase M2; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Gene Expression, Cell Culture, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: PKM2 activators, DASA-58 and TEPP-46, inhibit multiple signaling pathways in BMMs. BMMs were treated with PKM2 activators DASA-58 ( A ) and TEPP-46 ( B ), respectively, in parallel with RANKL induction, and proteins were extracted at the corresponding time points, showing their effects on P38, JNK, ERK, P65, IκB-α, and IKKα/β protein phosphorylation. DASA-58 and TEPP-46 were used at 10 mM in the culture medium. Relative quantification of P38, JNK, ERK, P65, IκB-α, and IKKα/β protein phosphorylation with PKM2 activators DASA-58 ( C ) and TEPP-46 ( D ). Data are expressed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. BMM, bone marrow–derived macrophage; ERK, extracellular signal–regulated kinase; JNK, c-Jun NH2-terminal kinase; PKM2, pyruvate kinase 2; RANKL, receptor activator of nuclear factor-κB ligand.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Protein-Protein interactions, Phospho-proteomics, Quantitative Proteomics, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: PKM2 regulates osteoclastogenesis by affecting osteoclast precursor cell fusion via downregulation of OC-STAMP and DC-STAMP

doi: 10.1016/j.jbc.2025.108439

Figure Lengend Snippet: PKM2 regulated osteoclastogenesis via inhibiting the expression of OC-STAMP and DC-STAMP. A , heatmap of part of the cluster analysis results of differentially expressed genes (threshold: log fold change <−1, p < 0.05). B , gene set enrichment analysis was used to select the involved pathways. C , OC-STAMP and DC-STAMP mRNA expression was inhibited after the knockdown of PKM2. D , OC-STAMP and DC-STAMP mRNA expression was suppressed after treatment with PKM2 activators, DASA-58 and TEPP-46. E , DC-STAMP protein expression was inhibited after the knockdown of PKM2. F , relative quantification of DC-STAMP protein. Data are displayed as the mean ± SD of three independent replicates. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. DC-STAMP, dendritic cell–specific transmembrane protein; OC-STAMP, osteoclast stimulatory transmembrane protein; PKM2, pyruvate kinase 2.

Article Snippet: Adenovirus carrying shRNA targeting murine PKM2 ( NM_011099 ) and control viruses were cloned and packaged by Vigene Biosciences.

Techniques: Expressing, Knockdown, Quantitative Proteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

doi: 10.1007/s00018-025-05688-8

Figure Lengend Snippet: Empagliflozin downregulated the mRNA level of PKM2 in DKD a Heatmap and volcano map of the identified differentially expressed genes (DEGs) b Bubble chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment for fibrosis-related pathways in Normal glucose (NG)-treated and high glucose (HG)-treated HK-2 cells. c Bubble chart of Gene Ontology (GO) enrichment for fibrosis-related pathways in Normal glucose (NG)-treated and high glucose (HG)-treated HK-2 cells. d KEGG analyses of enriched pathways upregulated in HG group and meanwhile downregulated by empagliflozin according to RNA-sequencing. e FPKM levels of glycolytic genes expression of glycolytic genes in HK-2 cells treated NG, HG or HG with empagliflozin according to RNA-sequencing. f Expression of glycolytic genes in HK-2 cells treated NG, HG or HG with empagliflozin analysed by RT-qPCR. g mRNA expression of PKM2 , PFKP , and HK2 in each CD-1 mouse group. h mRNA expression of PKM2 , PFKP , and HK2 in each C57BL/6 mouse group.For all panels, * p < 0.05. NG , normal glucose, HG , high glucose, EMPA , empagliflozin, CD , chow diet, HFD , high fat diet

Article Snippet: The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ , ].

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

doi: 10.1007/s00018-025-05688-8

Figure Lengend Snippet: Empagliflozin downregulated the protein level of PKM2 in DKD a Western blot analysis for PKM2 in HK-2 cells treated with NG, HG, or HG with empagliflozin. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. b Western blot analysis for PKM2 in each group of CD-1 mice. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. c Western blot analysis for PKM2 in each group of C57BL/6 mice. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. d – e Representative images of immunofluorescence staining for PKM2 and Lotus tetragonolobus lectin (LTL) in CD-1 and C57BL/6 mice. Scale bar, 100 μm. f Representative images of immunochemistry staining of PKM2 from kidney sections of CD-1 mice. Images were obtained under a light microscope at 100X lens. g Representative images of immunochemistry staining of PKM2 from kidney sections of C57BL/6 mice. Images were obtained under a light microscope at 100X lens. h Representative images of immunochemistry staining of PKM2 from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens. i – k Correlation analysis of the expression of PKM2 protein levels with COL3A1 and αSMA in kidneys of DKD patients, each group of CD-1 mice and each group or C57BL/6 mice. For all panels, * p < 0.05. NG normal glucose, HG high glucose, Ctrl control, CD chow diet, HFD high-fat diet, EMPA empagliflozin

Article Snippet: The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ , ].

Techniques: Western Blot, Immunofluorescence, Staining, Light Microscopy, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

doi: 10.1007/s00018-025-05688-8

Figure Lengend Snippet: Empagliflozin ameliorated renal fibrosis by downregulating PKM2 in vitro a – b Western blot analysis for PKM2 in HK-2 cells treated with empty vector or PKM2-overexpression plasmid (pcDNA3-PKM2). c – d Western blot analysis of PKM2 in HK-2 cells treated with shRNA-NC or shRNA-PKM2. e – g Western blot analysis of COL3A1 and αSMA in HK-2 cells treated with empty vector or pcDNA3-PKM2. h – k Western blot analysis of COL3A1, αSMA, and PKM2 in each group of HK-2 cells. l – o Western blot analysis of COL3A1, αSMA, and PKM2 in each group of HK-2 cells. For quantification, the band intensities of PKM2, COL3A1, and αSMA were normalized to respective band intensities of β-ACTIN

Article Snippet: The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ , ].

Techniques: In Vitro, Western Blot, Plasmid Preparation, Over Expression, shRNA

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

doi: 10.1007/s00018-025-05688-8

Figure Lengend Snippet: Empagliflozin ameliorated EMT in DKD a Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens. b Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of each group of CD-1 mice. Images were obtained under a light microscope at 100X lens. c Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of each group of C57BL/6 mice. Images were obtained under a light microscope at 100X lens. e – f Correlation analysis of the positive area of PKM2 with E-cadherin and Vimentin in kidneys of DKD patients, each group of CD-1 mice and C57BL/6 mice. For all panels, * p < 0.05. Ctrl control, CD chow diet, HFD high-fat diet, NG normal glucose, HG high glucose, EMPA empagliflozin, ECAD E-cadherin, VIM vimentin

Article Snippet: The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ , ].

Techniques: Staining, Light Microscopy, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

doi: 10.1007/s00018-025-05688-8

Figure Lengend Snippet: Empagliflozin downregulated PKM2 by blocking the binding of estrogen-related receptor alpha (ESRRA) to the promoter a Western blot analysis for cytoplasmic and nuclear ESRRA in kidneys of each CD-1 mouse group. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of LAMIN-B. b Western blot analysis for cytoplasmic and nuclear ESRRA in kidneys of each C57BL/6 mouse group. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of LAMIN-B. c Western blot analysis for cytoplasmic and nuclear ESRRA in HK-2 cells treated with NG, HG, or HG with empagliflozin. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of PCNA. d Representative images of intracellular immunofluorescence staining for ESRRA in HK-2 cells treated with NG, HG, or HG with empagliflozin. Scale bar, 100 μm. e Luciferase reporter assays for the PKM2 promoter were performed in HK-2 cells co-transfected with ESRRA expression plasmids and luciferase reporter plasmids containing wild-type or mutant mouse PKM2 promoters. f ChIP analysis for ESRRA binding to the promoter of PKM2 gene in HK-2 cells at different binding sites. g ChIP analysis for ESRRA binding to the promoter of PKM2 gene in HK-2 cells treated with NG, HG, or HG with Empagliflozin at different binding sites. For all panels, * p < 0.05. STZ streptozotocin, CD control diet, HFD high fat diet, NG normal glucose, HG high glucose, EMPA empagliflozin, DBS distant binding site, BS binding site

Article Snippet: The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ , ].

Techniques: Blocking Assay, Binding Assay, Western Blot, Immunofluorescence, Staining, Luciferase, Transfection, Expressing, Mutagenesis, Control