pkm2  (Proteintech)

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: GDF15 preserves mitochondrial homeostasis in LPS-stimulated macrophages through dual regulation of SMAD7 and PKM2 pathways. (A) HIF-1α and SMAD7 expression in RAW264.7 macrophages across conditions: Untreated, LPS, LPS with rAAV8-mGdf15 overexpression (LPS+GDF15), and LPS with GDF15 knockdown (si-GDF15). β-actin: loading control.(HIF-1α suppression and SMAD7 induction by GDF15.) (B) Cytosolic and nuclear PKM2 protein levels. Lamin B1 (nuclear) and α-tubulin (cytosolic) markers validate fractionation efficiency. Study groups and individual replicates are identified in the figure key.(PKM2 subcellular redistribution modulated by GDF15.) (C) Immunofluorescence of PKM2 (red) and nuclei (DAPI, blue). Arrows indicate nuclear PKM2 accumulation. Scale bar: 15 μm.(Nuclear PKM2 enrichment upon LPS challenge mitigated by GDF15 and exacerbated by GDF15 knockdown.).

Article Snippet: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

Techniques: Expressing, Over Expression, Knockdown, Control, Fractionation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: HIF-1α and PKM2 are critical effectors of GDF15-driven mitochondrial protection and anti-inflammatory responses. (A) HIF-1α inhibition by BAY 87-2243 (5 μM, 24 h). β-actin: loading control.(Pharmacological HIF-1α blockade.) (B) PKM2 inhibition by Shikonin (2 μM, 24 h). β-actin: loading control.(PKM2 activity suppression.) (C) UQCRC1 recovery in LPS-injured macrophages treated with: GDF15 overexpression, HIF-1α inhibitor (BAY), or PKM2 inhibitor (Shikonin). β-actin: loading control.(Mitochondrial complex III rescue via HIF-1α/PKM2 inhibition mirrors GDF15 effects.) (D) Inflammatory (TNF-α, IL-6) and metabolic (lactate) markers in cell supernatant (n = 5). Study groups and individual replicates are identified in the figure key. ***p < 0.001.(HIF-1α/PKM2 targeting replicates GDF15-mediated anti-inflammatory and metabolic homeostasis.) (E) UQCRC1 expression under GDF15 loss-of-function: si-GDF15 alone vs. combined with BAY 87–2243 or Shikonin. β-actin: loading control. Study groups and individual replicates are identified in the figure key.(Mitochondrial rescue in GDF15-deficient macrophages requires HIF-1α/PKM2 inhibition.) (F) Supernatant cytokines and lactate in si-GDF15 macrophages with/without inhibitors (n = 5). ***p < 0.001. (Inflammation reversal in GDF15-knockdown macrophages depends on HIF-1α/PKM2 blockade.).

Article Snippet: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

Techniques: Inhibition, Control, Activity Assay, Over Expression, Expressing, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: RPL35A drives aerobic glycolysis and tumorigenesis by facilitating MYC-mediated SKP2 transcription

doi: 10.1016/j.jbc.2025.110944

Figure Lengend Snippet: RPL35A knockdown suppresses glycolytic activity in lung cancer cells. A , gene set enrichment analysis (GSEA) revealed a positive correlation between RPL35A expression and aerobic glycolysis. B , Western blot analysis showed downregulation of key glycolytic enzymes (PKM2, HK2, GLUT1, ADH4, ALDOC) in A549 and H1299 cells following RPL35A knockdown. C–F , glucose consumption ( C ), lactate production ( D ), ATP levels ( E ), and extracellular acidification rate (ECAR), and ( F ) were significantly reduced upon RPL35A silencing, indicating impaired glycolysis. G , oxygen consumption rate (OCR) was increased after RPL35A knockdown, suggesting a shift toward oxidative phosphorylation. H–J , in RPL35A-overexpressing cells, treatment with the glycolysis inhibitor 2-DG reduced glucose uptake ( H ), lactate production ( I ), and ATP levels ( J ). K , 2-DG treatment attenuated the enhanced cell proliferation induced by RPL35A overexpression. L , 2-DG enhanced apoptosis in RPL35A-overexpressing cells. Data are presented as mean ± SD. Statistical significance: ∗ p < 0.05, p < 0.01, ∗ p < 0.001.

Article Snippet: Membranes were blocked for 1 h, then incubated overnight at 4°C with the following primary antibodies:RPL35A (Abcam, Rabbit, ab241070, 1:2000), MYC (CST, Rabbit, 5605, 1:1000), SKP2 (PTG, Rabbit, 15010-1-AP, 1:2000), GAPDH (Bioworld, Rabbit, AP0063, 1:3000), PKM2 (Bioss, Mouse, bs-0102M, 1:1000), HK2 (Proteintech, Rabbit, 66974-1-Ig, 1:3000), GLUT1 (Bioss, Rabbit, bs-4855R, 1:1000), ADH4 (Abcam, Rabbit, ab137077, 1:2000), ALDOC (Proteintech, Rabbit, 14884-1-AP, 1:5000), and Histone H3 (CST, Rabbit, 4499S, 1:2000).

Techniques: Knockdown, Activity Assay, Expressing, Western Blot, Phospho-proteomics, Over Expression

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing circGDI2 inhibits proliferation and glycolysis in HCC cells To explore the effect of circGDI2 on proliferation and glycolysis in HCC cells, sh-circGDI2 was transfected into Li-7 and Huh-7 cells. (A) The transfection efficiency was confirmed by RT-qPCR. (B) CCK-8 assay was used to assess cell proliferation. (C) Glucose consumption level and lactate production were detected using the Glucose Assay Kit with O-toluidine and Lactate Assay Kit. (D) RT-qPCR was used to analyze the expression changes of key glycolysis-related genes (HIF-1α. HK1, PKM2, PFKM, HK2, GLUT1, PFKP, LDHA) in Li-7 and Huh-7 cell lines after circGDI2 knockdown. (E) The expression level of PKM2 was examined in clinical HCC tissues and adjacent normal tissues using RT-qPCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group, or the Adjacent group.

Article Snippet: PKM2 , Western blot , Rabbit , 1:5000 , Proteintech , 15822-1-AP.

Techniques: Transfection, Quantitative RT-PCR, CCK-8 Assay, Glucose Assay, Lactate Assay, Expressing, Knockdown

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing circGDI2 inhibits proliferation and glycolysis and PKM2 expression through IGF2BP2 in HCC cells. (A) Western blot was used to analyze IGF2BP2 expression in Li-7 and Huh-7 cell lines after circGDI2 knockdown. (B) The expression level of IGF2BP2 was examined in clinical HCC tissues and adjacent normal tissues using RT-qPCR. To clarify if circGDI2 regulated HCC cell proliferation and glycolysis through IGF2BP2, sh-circGDI2 and OE-IGF2BP2 were co-transfected into Li-7 and Huh-7 cells. (C) Western blot was used to assess the effect of IGF2BP2 on the expression of PKM2. (D) CCK-8 assay was used to assess cell proliferation. (E) Glucose consumption level and lactate production were detected using the Glucose Assay Kit with O-toluidine and Lactate Assay Kit. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group, or the Adjacent group. # P < 0.05, ## P < 0.01, ### P < 0.001, vs. the sh-circGDI2+OE-NC group.

Article Snippet: PKM2 , Western blot , Rabbit , 1:5000 , Proteintech , 15822-1-AP.

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Glucose Assay, Lactate Assay

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing circGDI2 inhibits HCC tumor growth and PKM2 expression through IGF2BP2. To verify the effect of circGDI2 and IGF2BP2 on HCC tumor growth, a xenograft mouse model was constructed. (A) Pictures of the isolated tumors of the indicated group. (B) The tumor volume and weight were recorded. (C) HE staining, Tunel and Ki-67 staining were performed to observe the histological characteristics and cell proliferation in the tumor tissues (scale bar = 100 μm). (D) RT-qPCR was used to detect circGDI2, IGF2BP2 and PKM2 levels. (E) IHC was used to detect IGF2BP2 and PKM2 levels (scale bar = 100 μm). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, vs. the sh-circGDI2+OE-NC group.

Article Snippet: PKM2 , Western blot , Rabbit , 1:5000 , Proteintech , 15822-1-AP.

Techniques: Expressing, Construct, Isolation, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing FTO inhibits HCC tumor growth and decreases circRNA, IGF2BP2 and PKM2 levels. To investigate the biological role of FTO on HCC tumor growth, the xenograft tumor models of HCC cells in the sh-NC and sh-FTO groups were established. (A) Pictures of the isolated tumors of the indicated group. (B) The tumor volume and weight were recorded. (C) HE staining, Ki-67 staining and Tunel stainning were performed to observe the histological characteristics and cell proliferation in the tumor tissues (scale bar = 100 μm). (D) RT-qPCR was used to detect circGDI2 and FTO levels. (E) IHC was used to detect IGF2BP2 and PKM2 levels (scale bar = 100 μm). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group.

Article Snippet: PKM2 , Western blot , Rabbit , 1:5000 , Proteintech , 15822-1-AP.

Techniques: Isolation, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: PKM2 inhibitor suppresses kidney fibrogenesis by disrupting YAP-TEAD-CCN2 transcriptional signaling following ischemia–reperfusion injury

doi: 10.1016/j.jbc.2025.111029

Figure Lengend Snippet: Evaluation of renal fibrosis using Masson's trichrome and PKM2 immunofluorescence staining . A , representative images of kidney sections stained with Masson’s trichrome 14 days following UIRI. This scale bar represents 500 μm. B , didney sections stained with anti-PKM2 antibody (Alexa Fluor 555: red ) and Lotus tetragonolobus lectin (LTL; FITC: green ). The cell nuclei were counterstained with DAPI ( blue ) (N = 6). This scale bar represents: 100 μm. Data were acquired using a confocal laser scanning microscope. LTL, Lotus tetragonolobus lectin; UIRI, unilateral ischemia–reperfusion injury.

Article Snippet: The sections were then blocked with 5% skim milk in PBS for 5 min. To detect PKM2, the sections were incubated with a primary antibody against PKM2 (rabbit anti-PKM2, 800 μg/ml, #15822-1-AP; Proteintech) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 4 °C for 24 h. The primary antibody was visualized using Alexa Fluor 555-conjugated secondary antibody (goat anti-rabbit IgG, #A21428; Molecular Probes, Thermo Fisher Scientific) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 20 °C–25 °C (room temperature, RT) for 1 h. Rabbit IgG, diluted 1:375 (1 mg/ml, #30000-0-AP; Proteintech), was used as a negative control.

Techniques: Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: The Journal of Biological Chemistry

Article Title: PKM2 inhibitor suppresses kidney fibrogenesis by disrupting YAP-TEAD-CCN2 transcriptional signaling following ischemia–reperfusion injury

doi: 10.1016/j.jbc.2025.111029

Figure Lengend Snippet: In vitro analysis of 3k treatment and siPKM2 transfection in a human renal tubular epithelial (HK-2) cell injury model . A , time course analysis of the effects of oxygen–glucose deprivation and reoxygenation (OGD/R) and 3k treatment. mRNA expression analysis of PKM2 , TEAD2 , and CCN2 in ( B ) 3k-treated and ( C ) siPKM2-transfected cells, from 24 to 72 h. Statistical comparisons between the control or the mock and treatment groups at each time point were performed using the Wilcoxon test. ∗ indicates statistical significance ( p < 0.05). Individual data points represent the median and IQR (N = 6).

Article Snippet: The sections were then blocked with 5% skim milk in PBS for 5 min. To detect PKM2, the sections were incubated with a primary antibody against PKM2 (rabbit anti-PKM2, 800 μg/ml, #15822-1-AP; Proteintech) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 4 °C for 24 h. The primary antibody was visualized using Alexa Fluor 555-conjugated secondary antibody (goat anti-rabbit IgG, #A21428; Molecular Probes, Thermo Fisher Scientific) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 20 °C–25 °C (room temperature, RT) for 1 h. Rabbit IgG, diluted 1:375 (1 mg/ml, #30000-0-AP; Proteintech), was used as a negative control.

Techniques: In Vitro, Transfection, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: PKM2 inhibitor suppresses kidney fibrogenesis by disrupting YAP-TEAD-CCN2 transcriptional signaling following ischemia–reperfusion injury

doi: 10.1016/j.jbc.2025.111029

Figure Lengend Snippet: Western blot analysis of cross-linked protein expression and Co-IP analysis in HK-2 cells . A , western blot analysis of cross-linked proteins extracted from 3k-treated HK-2 cells. B and C , Co-IP analysis of the interaction between YAP1, β-catenin, and PKM2 in HK-2 cells. IP was performed using protein samples from HK-2 cells overexpressing CCN2. Co-IP with an anti-PKM2 antibody confirmed that both β-catenin and YAP1 co-precipitated with PKM2 (N = 6), ( D ) as illustrated in the schematic diagram. CCN2, cellular communication network factor 2.

Article Snippet: The sections were then blocked with 5% skim milk in PBS for 5 min. To detect PKM2, the sections were incubated with a primary antibody against PKM2 (rabbit anti-PKM2, 800 μg/ml, #15822-1-AP; Proteintech) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 4 °C for 24 h. The primary antibody was visualized using Alexa Fluor 555-conjugated secondary antibody (goat anti-rabbit IgG, #A21428; Molecular Probes, Thermo Fisher Scientific) diluted 1:300 in blocking buffer (5% skim milk/PBS) at 20 °C–25 °C (room temperature, RT) for 1 h. Rabbit IgG, diluted 1:375 (1 mg/ml, #30000-0-AP; Proteintech), was used as a negative control.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay