Journal: International Journal of Biological Sciences

Article Title: Atg7 inhibits Warburg effect by suppressing PKM2 phosphorylation resulting reduced epithelial-mesenchymal transition

doi: 10.7150/ijbs.26077

Figure Lengend Snippet: Atg7 interacts with PKM2 directly and inhibits PKM2 phosphorylation. Atg7 interacts with PKM2. (A, B) Coimmunoprecipitation (IP) of endogenous Atg7 with PKM2 in HeLa cells. Cell lysates were subjected to IP using anti-PKM2(rabbit) or anti-Atg7(rabbit) and unrelated rabbit IgG as a control. The resulting precipitates were subjected to Western blot (WB) analysis with anti-Atg7 or anti-PKM2. A portion of the whole-cell lysate (WCL) of the input for IP was also subjected to IB analysis. (C) HEK293T cells were co-transfected with expression plasmids encoding Myc-tag or Myc-tagged Atg7 and Flag-tagged PKM2 as indicated. Cells were lysed and subjected to IP with an anti-Myc antibody. The resulting precipitates were subjected to WB analysis with anti-Flag antibody. A portion of the whole-cell lysate of the input for IP was also subjected to IB analysis. (D, E) GST pull-down assay was performed with glutathione S-transferase (GST) or GST fused PKM2 protein and in vitro translated Flag-Atg7 (D) or GST fused Atg7 protein and in vitro translated Flag-PKM2 (E). Input and pull-down fractions of Flag-Atg7 (D) or Flag-PKM2 (E) were detected by immunoblots using anti-Flag antibodies. The corresponding SDS-PAGE gels show the amount of GST or GST-PKM2/Atg7 immobilized in each assay. Atg7 inhibitsTyr-105 phosphorylation of PKM2. (F) Tissues (brain, kidney, liver and lung) from Atg7 knockout mice and wide type mice were gained soon after born and tissue lysates was used for Western blot assay to detect total and Tyr-105 phosphorylation of PKM2. (G) Western blot analysis of total and Tyr-105 phosphorylation of PKM2 in wide type and Atg7 knockout MEF cells.

Article Snippet: The PKM2 expression plasmid of was bought from OriGene (SC315792).

Techniques: Western Blot, Transfection, Expressing, Pull Down Assay, In Vitro, SDS Page, Knock-Out

Journal: International Journal of Biological Sciences

Article Title: Atg7 inhibits Warburg effect by suppressing PKM2 phosphorylation resulting reduced epithelial-mesenchymal transition

doi: 10.7150/ijbs.26077

Figure Lengend Snippet: Atg7 inhibits PKM2 phosphorylation via blocking the interaction of PKM2 and FGFR1. Atg7 inhibitsTyr-105 phosphorylation of PKM2. (A, B, C) Western blot analysis of total and Tyr-105 phosphorylation of PKM2 in HeLa cell expressing Myc-Atg7(A), shRNA-Atg7(B), HeLa cell stable knockdown of endogenous PKM2 and “rescue” expression of Myc-Atg7 NTm (nontargetable mutant). (C) and their corresponding control cells. Right: Quantification of protein expression (Tyr-105 phosphorylation of PKM2) shown in (A, B, C) normalized to β-actin. Data shown are mean±S.E.M. of n≥3 technical replicates and are representative of three independent experiments. P values were calculated by t-test. *P<0.005, **P<0.001. Atg7 blocked the interaction of PKM2 and FGFR1(D, E). Coimmunoprecipitation (IP) of endogenous PKM2 with FGFR1 in HeLa cell transfected with expression plasmids encoding Myc-tag or Myc-tagged Atg7(D), HeLa cell stable knockdown of endogenous PKM2(E). Cell lysates were subjected to IP using anti-FGFR1(rabbit) and unrelated rabbit IgG as a control. The resulting precipitates were subjected to WB analysis with anti-PKM2. A portion of the whole-cell lysate (WCL) of the input for IP was also subjected to IB analysis. (F) HeLa cell stable knockdown of endogenous PKM2 and control cell were treated with bFGF(10 ng/mL), cell lysates were subjected to IP using anti-FGFR1(rabbit). The resulting precipitates were subjected to Immunoblot (IB) analysis with anti-PKM2. A portion of the whole-cell lysate (WCL) of the input for IP was also subjected to WB analysis.

Article Snippet: The PKM2 expression plasmid of was bought from OriGene (SC315792).

Techniques: Blocking Assay, Western Blot, Expressing, shRNA, Mutagenesis, Transfection

Journal: International Journal of Biological Sciences

Article Title: Atg7 inhibits Warburg effect by suppressing PKM2 phosphorylation resulting reduced epithelial-mesenchymal transition

doi: 10.7150/ijbs.26077

Figure Lengend Snippet: Atg7 inhibition promotes epithelial-mesenchymal transition through enhanced Warburg effect. Mutational analysis revealed that substitution of Y105 to E105 results in a significant increase glucose consumption(A), lactate production(B) in Atg7 knockdown cells. P values were calculated by One Way ANOVA. *P<0.005, **P<0.001. (C) Western blot analysis of EMT markers in knockdown Atg7 and PKM2. (D) 2DG (inhibitor of glycolysis) administration in Atg7-knockdown cell, Western blot analysis of EMT markers in HCT-116, HeLa and MEF cells. (E) Transwell assay in HCT-116 2DG administration. Original magnification, ×200. Scale bar represents 100 μm. (F) Statistical analysis result of transwell assay in (E). P values were calculated by One Way ANOVA. *P<0.005, **P<0.001.

Article Snippet: The PKM2 expression plasmid of was bought from OriGene (SC315792).

Techniques: Inhibition, Western Blot, Transwell Assay

Journal: International Journal of Biological Sciences

Article Title: Atg7 inhibits Warburg effect by suppressing PKM2 phosphorylation resulting reduced epithelial-mesenchymal transition

doi: 10.7150/ijbs.26077

Figure Lengend Snippet: Model for Atg7 inhibiting Warburg effect by suppressing PKM2 phosphorylation resulting reduced EMT. Atg7 interacts with PKM2 directly and inhibits PKM2 phosphorylation. PKM2 dephosphorylation inhibits the Warburg effect, thereby inhibiting EMT. See text for details.

Article Snippet: The PKM2 expression plasmid of was bought from OriGene (SC315792).

Techniques: De-Phosphorylation Assay

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: The Sequences of the Pkm2-PACH

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Sequencing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: Rat Primer Sequences for qRT-PCR

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Sequencing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: Human Primer Sequences for qRT-PCR

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Sequencing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: The effects of CSE on mitochondrial oxidative damage and Pkm2/Nrf2 signaling pathway in A549 cells. ( A and C ) The effects of 10%CSE induction for 6 hours, 12 hours, and 24 hours on the generation of mROS in A549 cells. ( B ) Inhibition of mitochondrial membrane potential after 10%CSE induction for 6 hours, 12 hours, and 24 hours. ( D and E ) Inhibition of mitochondrial respiratory chain complex I and III after 10%CSE induction for 6 hours, 12 hours, and 24 hours. ( F and G ) Inhibition of T-SOD and GSH-Px after 10%CSE induction for 6 hours, 12 hours, and 24 hours. ( H ) Western blot analysis for assessing the effects of mitochondrial-related pathway including caspase-3, Bcl-2, Bax, and Cyt-C in A549 cells following 10%CSE induction. ( I ) Quantitative real-time PCR analysis for assessing the effects of Pkm2/Nrf2 signaling pathway including Pkm2, Nrf2, GCLC, and GCLM in A549 cells following 10%CSE induction. ( J ) Western blot analysis for assessing the effects of Pkm2/Nrf2 signaling pathway including Pkm2, Nrf2, GCLC, and GCLM in A549 cells following 10%CSE induction. All data are represented as mean ± SD (n=3). * P < 0.05 and ** P < 0.01 as compared to Con group, # P < 0.05 and ## P < 0.01 as compared to 10%CSE-6h group, and ■ P < 0.05 and ■■ P < 0.01 as compared to 10%CSE-12h group.

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Membrane, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: Mitochondria can be protected from oxidative damage by activating the Pkm2/Nrf2 signaling pathway. ( A (i) and (ii)) The production of mtROS after treated for Pkm2 expression plasmid in A549 cells. ( B – D ) Changes in mitochondrial membrane potential, and enzymatic activities of mitochondrial respiratory chain complexes I and III. ( E and F ) The activity of T-SOD and GSH-Px with Pkm2 expression plasmid. ( G ) Western blot analysis for assessing the effects of Pkm2/Nrf2 signaling pathway including Pkm2, Nrf2, GCLC, and GCLM in A549 cells following Pkm2 expression plasmid transfection. All data are represented as mean ± SD (n=3). * and ** indicate P < 0.05 and P < 0.01.

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Membrane, Activity Assay, Western Blot, Transfection

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: Effects of ECC-BYF III on Pkm2/Nrf2 signaling pathway in lung tissue of COPD rats. ( A – D ) Pkm2, Nrf2, GCLC and GCLM mRNA expression in lung tissue; ( E – I ) Pkm2, Nrf2, GCLC and GCLM levels as assessed by Western blot. All data are represented as mean ± SD (n=3). * P < 0.05 and ** P < 0.01 as compared to Normal group, # P < 0.05 and ## P < 0.01 as compared to Model group.

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway

doi: 10.2147/COPD.S468825

Figure Lengend Snippet: Schematic diagram of ECC-BYF III regulates Pkm2/Nrf2 signaling pathway.

Article Snippet: The Pkm2 expression plasmid was synthesized by Sangon Biotech (Shanghai, China).

Techniques:

Journal: Cell Death & Disease

Article Title: YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis

doi: 10.1038/s41419-022-04711-1

Figure Lengend Snippet: A RT-PCR analysis on the correlation between YTHDF1 and PKM2 mRNA. B The m6A modification motif of PKM2 mRNA for YTHDF1 binding, analyzed by RMBase V2.0 database ( http://rna.sysu.edu.cn/rmbase/ ). C Breast cancer cell lines were co-transfected with siYTHDF1 and pmirGLO-PKM2 reporter for 48 h to determine the translation efficiency of PKM2 after different treatment, which was calculated by dividing protein yield with mRNA abundance (F-luc/R-luc). D YTHDF1 and PKM2 expression levels in breast cancer cells after the co-transfection with YTHDF1 siRNA and pCDNA3.1-PKM2-3×FLAG. E pH changes in the culture medium of breast cancer cells in different groups after 48 h. F , G The concentration of glucose and lactic acid in the culture medium of the breast cancer cells in different groups after 48 h. H Detection of YTHDF1 and PKM2 expression in the breast cancer cells after co-transfection with pCDNA3.1-YTHDF1-3×FLAG and PKM2 siRNA. I pH changes in the culture medium of breast cancer cells in different groups after 48 h. J , K The concentration of glucose and lactate in the culture medium of the breast cancer cells after 48 h. L Apoptosis levels of breast cancer cells after co-transfection with YTHDF1 siRNA and pCDNA3.1-PKM2-3×FLAG via FACS. M Apoptosis levels of breast cancer cells after co-transfection with pCDNA3.1-YTHDF1-3×FLAG and PKM2 siRNA via FACS. Statistical analysis results are presented as mean ± SEM, student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human YTHDF1 and PKM2 overexpression vectors were constructed by inserting YTHDF1 (NM_017798.4) and PKM2 (NM_002654.6) into pCDNA3.1-3×FLAG (Addgene, #53556) plasmid respectively, of which the sequences were synthesized by Miaolingbio Ptd Ltd.

Techniques: Reverse Transcription Polymerase Chain Reaction, Modification, Binding Assay, Transfection, Expressing, Cotransfection, Concentration Assay

Journal: Cell Death & Disease

Article Title: YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis

doi: 10.1038/s41419-022-04711-1

Figure Lengend Snippet: A , B Impact of YTHDF1 inhibition on the growth of subcutaneous tumors. C Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors at 28 days after transplantation. D , E Impact of YTHDF1 overexpression on the growth of subcutaneous tumors. F Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors. G , H Impact of agomir-16-5p transfection on the growth of subcutaneous tumors. I Western blot analysis regarding YTHDF1 and PKM2 expression in subcutaneous tumors at 24 days after agomir-16-5p treatment. J – L Immunohistochemical image regarding the lung metastasis of breast tumors in mice after YTHDF1 knockdown, YTHDF1 overexpression or agomir-16-5p treatment. M Immunohistochemical imaging of YTHDF1 and PKM2 in matched normal and cancerous tissues of breast cancer patients. N Schematic illustration showing YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis.

Article Snippet: Human YTHDF1 and PKM2 overexpression vectors were constructed by inserting YTHDF1 (NM_017798.4) and PKM2 (NM_002654.6) into pCDNA3.1-3×FLAG (Addgene, #53556) plasmid respectively, of which the sequences were synthesized by Miaolingbio Ptd Ltd.

Techniques: Inhibition, Western Blot, Expressing, Transplantation Assay, Over Expression, Transfection, Immunohistochemical staining, Knockdown, Imaging

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: Clinical correlation of HSP90 and PKM2 expression in HCC

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Expressing

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 bound to PKM2 and increased the abundance of PKM2 protein in HCC cells. a Flag-HSP90 and HA-PKM2 plasmids were transfected into HEK293 cells. Immunoprecipitation was performed using anti-Flag or anti-HA antibody. HSP90 and PKM2 showed an interaction between each other in HEK293 cells. b The interaction between endogenous HSP90 and PKM2 proteins in Hep3B cells was analyzed by Co-IP. Endogenous HSP90 and PKM2 proteins interacted with each other in Hep3B cells. c GST pull-down assays were performed to investigate the direct interaction between HSP90 and PKM2. d Hep3B cells were transfected with HSP90 shRNA or negative-control (NC) shRNA. 72 h after transfection, the levels of HSP90 and PKM2 protein in Hep3B cells were examined. Knockdown of HSP90 decreased PKM2 protein in Hep3B cells. e Huh7 cells were transduced with retroviruses encoding Flag-HSP90 or empty control retroviruses. 72 h after viral transduction, the levels of HSP90 and PKM2 protein in Huh7 cells were examined. Forced expression of HSP90 increased PKM2 protein abundance in Huh7 cells. *, P < 0.05 by t-test

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, shRNA, Negative Control, Transduction, Expressing

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 expression was positively correlated with PKM2 expression in HCC tissues. a IHC staining of HSP90 and PKM2 in HCC tissues and the adjacent non-tumor tissues were performed. HSP90 and PKM2 expression in HCC tissues were increased compared with non-tumor tissues. HCC tissues with high HSP90 level showed increased PKM2 protein level compared with those with low HSP90 level. b Compared with tissues with negative HSP90 expression, positive rate of PKM2 was significantly increased in HCC tissues with positive HSP90 expression. c HSP90 IHC scores in HCC tissues were positively correlated with the IHC scores of PKM2. *, P < 0.05 by Chi-square test and Pearson correlation analysis

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 regulated the stability of the PKM2 protein in HCC cells. a Huh7 cells were transfected with empty vector or Flag-HSP90 vector. Hep3B cells were transfected with negative control (NC) shRNA or HSP90 shRNA. qRT-PCR demonstrated that neither HSP90 overexpression or knockdown changed the mRNA level of PKM2. b & c The protein half-life of PKM2 in Hep3B cells was analyzed following treatment with cycloheximide (CHX). The PKM2 turnover rate was lower in Hep3B cells with HSP90 knockdown and it was higher in HSP90 overexpressing Huh7 cells. d MG-132 was used to inhibit the proteasomal degradation in Hep3B cells. MG-132 treatment reversed the downregulation of PKM2 protein induced by HSP90 knockdown. *, P < 0.05 by one way ANOVA test

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Quantitative RT-PCR, Over Expression

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 increased PKM2 phosphorylation at Thr-328. a HA-tagged PKM2 was co-transfected with empty vector or Flag-tagged HSP90 in HEK293 cells. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HA and Flag. Forced expression of Flag-tagged HSP90 in HEK293 cells increased the phosphorylation of HA-tagged PKM2. b Huh7 cells were transfected with empty vector or Flag-tagged HSP90. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HSP90 and GAPDH. Overexpression of HSP90 increased PKM2 Ser/Thr phosphorylation in Huh7 cells. c Hep3B cells were transfected with negative control (NC) shRNA or HSP90 shRNA. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HSP90 and GAPDH. Knockdown of HSP90 decreased PKM2 Ser/Thr phosphorylation in Hep3B cells. d Phosphor-specific protein enrichment and MS identified two putative phosphorylation sites in PKM2 regulated by HSP90. S405 and T326 residues were highlighted by red. e Huh7 cells were transfected with empty vector or Flag HSP90, along with PKM2-S405A mutant (Serine 405 to alanine mutation) or HA-T328A mutant (Threonine 328 to alanine mutation). T328A mutant, instead of S405A, abrogated the increased phosphorylation of PKM2 induced by HSP90 overexpression. f Huh7 cells were co-transfected with empty vector or Flag-HSP90, and, HA-tagged wild type (WT) PKM2 or HA-tagged T328A mutant PKM2. The protein half-life of HA-tagged WT or mutated PKM2 was analyzed following treatment with cycloheximide (CHX). HSP90 overexpression increased the half-life of the wild type PKM2 while failed to increase the half time of T328A PKM2 mutant

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Over Expression, Negative Control, shRNA, Protein Enrichment, Mutagenesis

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 increases PKM2 phosphorylation through GSK-3β. a Co-IP was performed for HSP90, PKM2 and GSK-3β to confirm whether these protein formed protein complex in Huh7 cells. b Huh7 cells transfected with HSP90 vector or control vector were treated with GSK3i IX, a GSK-3β inhibitor. Phosphorylation of PKM2 was examined by western blot after inhibiting GSK-3β activity. c Huh7 cells transfected with HSP90 vector or control vector were treated with GSK-3β siRNA to knockdown GSK-3β. Phosphorylation of PKM2 was examined by western blot after GSK-3β knockdown. d In vitro kinase assay to determine the effects of recombinant GSK3β on threonine phosphorylation of PKM2-WT or T328A

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Activity Assay, In Vitro, Kinase Assay, Recombinant

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: Thr-328 phosphorylation was required for the functional influence of PKM2 on glycolysis, proliferation and apoptosis of HCC cells. Compared with Huh7 cells transfected with wild-type PKM2, T328A PKM2 mutant significantly reduced ( a ) glucose consumption, ( b ) lactate production and ( c ) PK catalytic activity of Huh7 cells. Compared with Huh7 cells transfected with wild-type PKM2, Thr-328A mutated PKM2 significantly decreased the ( d ) proliferation and increased the ( e ) Caspase-3 activity and ( f ) apoptosis the of Huh7 cells. *, P < 0.05 by t test

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Functional Assay, Transfection, Mutagenesis, Activity Assay

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 enhanced the glycolysis and proliferation while decreased the apoptosis of HCC cells through PKM2. Huh7 cells were co-transfected with corresponding vectors. Overexpression of HSP90 significantly increased ( a ) glucose consumption, ( b ) lactate production and ( c ) PK activity of Huh7 cells. PKM2 knockdown abrogated the promoting effects of HSP90 on glycolysis. Furthermore, overexpression of HSP90 significantly ( d ) increased proliferation while decreased the ( e ) Caspase-3 activity and ( f ) apoptosis of Huh7 cells. PKM2 knockdown abrogated the regulatory effects of HSP90 overexpression on cell proliferation and apoptosis. *, P < 0.05 by t test

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Transfection, Over Expression, Activity Assay

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: HSP90 promoted the growth of Huh7 cells through PKM2 in vivo. a Subcutaneous injection was performed using Huh7 cells co-transfected with corresponding vectors. Overexpression of HSP90 significantly increased the growth of Huh7 cells in nude mice while PKM2 knockdown abrogated the promoting effects of HSP90 on the growth of Huh7 cells in vivo. *, P < 0.05 by two-way ANOVA. b Tumor nodules were subjected to IHC staining for Ki-67, TUNEL assays and quantitative analysis. IHC staining for Ki67 and TUNEL assays revealed that HSP90 overexpression significantly increased the number of Ki-67 positive cells and decreased the number of apoptotic cells. These effects induced by HSP90 overexpression were abrogated by PKM2 knockdown. *, P < 0.05 by One-way ANOVA

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: In Vivo, Injection, Transfection, Over Expression, Immunohistochemistry, TUNEL Assay

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: The prognostic value of HSP90 and PKM2 for HCC patients. HCC patients were divided into HSP90 positive group and HSP90 negative group based on IHC staining. Patients with positive expression of HSP90 had significant shorter ( a ) overall survival (OS) and ( b ) disease free survival (DFS). HCC patients were divided into PKM2 positive group and PKM2 negative group based on IHC staining. Patients with positive expression of PKM2 had obvious poorer ( c ) OS and ( d ) DFS. HCC patients were divided into four groups: HSP90 postive PKM2 postive group, HSP90 postive PKM2 negative group, HSP90 negative PKM2 postive group and HSP90 negative PKM2 negative group. HCC patients in HSP90 negative PKM2 negative group had the best ( e ) OS and ( f ) DFS while those in HSP90 postive PKM2 postive group had the lowest ( e ) OS and ( f ) DFS. *, P < 0.05 by Log-rank test

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Immunohistochemistry, Expressing

Journal: Molecular Cancer

Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

doi: 10.1186/s12943-017-0748-y

Figure Lengend Snippet: Purification of PKM2- and HSP90-associated proteins

Article Snippet: Vector pLKO was purchased from Addgene (Cambridge, MA, USA) for the expression of PKM2 shRNA as previously described [ ].

Techniques: Purification

Journal: Nature Communications

Article Title: PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation

doi: 10.1038/ncomms8882

Figure Lengend Snippet: ( a ) Pyruvate kinase (PK) enzymatic activity in lysates of Huh7, Hep3B and Snu-449 cells stably expressing shNS or shPARP14. WBs showing the levels of PARP14 and PKM2 proteins in matching cell lysates. ( b ) Quantified intracellular pyruvate concentrations in control and shPARP14 HCC cells (Huh7 and Hep3B cells). ( c ) PK enzymatic activity in lysates of Hep3B cells left untreated (ctr.) or treated with 10 μM PJ-34 for 48 h. WBs showing the levels of endogenous PKM2 in matching cell lysates. ( d ) Glucose consumption and lactate production in Hep3B cells left untreated (ctr.) or treated with 10 μM PJ-34 for 48 h. ( e ) PK enzymatic activity in PARP14-depleted HCC cells co-expressing either PKM2 (shPARP14/shPKM2) or control NS (shPARP14/shNS) shRNAs. WBs analyses with antibodies against endogenous (endog.) proteins in co-silenced HCC cells used for the corresponding assay. Lysates of HEK293T cells overexpressing FLAG-PKM1 or FLAG-PKM2 were used as positive controls (pos. ctr). ( f ) Glucose consumption and lactate production in Huh7 and Snu-449 co-expressing shPARP14/shNS or shPARP14/shPKM2. ( g ) PK enzymatic activity and lactate production in Hep3B cells stably expressing FLAG-PKM1 (pWPI-FLAG-PKM1) or control empty vector (pWPI). WBs showing the levels of endogenous PKM2 and exogenous PKM1 in cell lysates. ( h ) Growth curves of Hep3B cells stably expressing FLAG-PKM1 (pWPI-FLAG-PKM1) or control empty vector (pWPI). ( a – h ) Data shown are mean±s.e.m. of n ≥3 technical replicates and are representative of at least three independent experiments. P values were calculated by Student's t -test.

Article Snippet: The full-length complementary DNAs of human PKM2 and PKM1 were obtained from pWZL-FLAG-PKM2 (Addgene plasmid 20585) and pET28-hPKM1 (Addgene plasmid 44241) , respectively, and then cloned between BamHI and XhoI sites of either pcDNA-HA- or pcDNA-FLAG-expressing vectors. pcDNA-HA-PKM2 mutant forms (S362A and T365A) were generated using the QuickChange XL site-directed mutagenesis kit (Stratagene).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation

doi: 10.1038/ncomms8882

Figure Lengend Snippet: ( a – d ) Analyses of shNS- and shPARP14-expressing Hep3B and Snu-449 cells cultured at the indicated time under normal ( a , b ) or hypoxic conditions ( c , d ) showing the percentage of apoptosis in the population sub-G1 (DNA content) ( a , c ) and the levels of apoptotic markers ( b , d ) by WBs. Closed and open arrowheads indicate the pro-cleaved and cleaved (active) products of the indicated proteins, respectively. p54 and p46 denote the JNK splicing isoforms. In vitro JNK1 kinase activity (JNK1 KA) was performed using glutathione S-transferase c-Jun as substrate in the presence of [32P]-γ-ATP. ( e ) Morphological assessment of apoptosis by electron microscopy in Hep3B cells expressing control shNS versus shPARP14. shNS-expressing Hep3B cells exhibit an homogenous nuclear envelop (NE), distinct nucleoli (N), euchromatin (E) associated with the nuclear pores (P), normal distribution of endoplasmic reticulum (ER) and mitochondria (M). shPARP14-expressing Hep3B cells displays early signs of apoptosis, such as clear structural alteration of the shape and surface of the cells, cytoplasmic disorganization (CD) with the presence of poorly defined ER and organelles, non-homogenous NE and peripheral membrane blebs (MB). Scale bar, 2 μm. ( f ) Percentage of apoptosis in PARP14-depleted HCC cells co-expressing either shPKM2 or HA-PKM2. WBs analyses showing endogenous (endog.) and ectopic expression of indicated proteins. ( a , c , f ) Data shown are mean±s.e.m. of three independent cultures. P values were calculated by Student's t -test.

Article Snippet: The full-length complementary DNAs of human PKM2 and PKM1 were obtained from pWZL-FLAG-PKM2 (Addgene plasmid 20585) and pET28-hPKM1 (Addgene plasmid 44241) , respectively, and then cloned between BamHI and XhoI sites of either pcDNA-HA- or pcDNA-FLAG-expressing vectors. pcDNA-HA-PKM2 mutant forms (S362A and T365A) were generated using the QuickChange XL site-directed mutagenesis kit (Stratagene).

Techniques: Expressing, Cell Culture, In Vitro, Activity Assay, Electron Microscopy, Control, Membrane

Journal: Nature Communications

Article Title: PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation

doi: 10.1038/ncomms8882

Figure Lengend Snippet: ( a , b ) PKM2 activity was assessed in lysates of HEK293T cells transfected with HA-PKM2 (10 μg) in combination with increasing amounts of constitutive active JNK1 (JNK1 CA ; 2.5, 5, 10 and 20 μg) or control empty vector (−) ( a ) or with FLAG-PKM2 (15 μg) and non-active HA-JNK1 (15 μg) ( b ). Lysates of HEK293T cells overexpressing JNK1 CA were used as positive controls (pos. ctr) for the detection of phospho-active JNK (p-JNK). WB analyses showing levels of phosphorylated endogenous (endog.) JNK1 and JNK1 CA (MKK7-JNK1α1), and HA and PKM2. Levels of total JNK1 serve as loading control. ( c ) PKM1 enzymatic activity was assessed in lysates of HEK293T cells transfected with equal amounts of HA-PKM1 (15 μg) and JNK1 constitutive active (JNK1 CA ; 15 μg) or control empty vector (−; 15 μg). WBs analyses showing levels of phosphorylated endogenous (endog.) JNK1 and JNK1 CA (MKK7-JNK1α1), and HA and PKM1. ( a – c ) Data shown are mean±s.e.m. of three biological replicates for all assays. P values were calculated by one-way analysis of variance ( P <0.0001) followed by Bonferroni's multiple comparison tests.

Article Snippet: The full-length complementary DNAs of human PKM2 and PKM1 were obtained from pWZL-FLAG-PKM2 (Addgene plasmid 20585) and pET28-hPKM1 (Addgene plasmid 44241) , respectively, and then cloned between BamHI and XhoI sites of either pcDNA-HA- or pcDNA-FLAG-expressing vectors. pcDNA-HA-PKM2 mutant forms (S362A and T365A) were generated using the QuickChange XL site-directed mutagenesis kit (Stratagene).

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Comparison

Journal: Nature Communications

Article Title: PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation

doi: 10.1038/ncomms8882

Figure Lengend Snippet: ( a ) Protein lysates of HEK293T cells transfected with FLAG-PKM2 in combination with HA-JNK1, HA-JNK2 or empty vector were subject to immunoprecipitation (IP) with anti-FLAG antibody followed by WBs analyses as indicated. ( b ) Activated JNK1 was immunoprecipitated (IP:JNK1 and WB:p-JNK) from lysates of Huh7 and PLC5 cells expressing nonspecific (shNS) or PARP14 (shPARP14) shRNAs and assayed for kinase activity (KA) using recombinant (rec.) His-PKM2 as substrate in the presence of [32P]-γ-ATP. [32P]-PKM2 (endog.) denotes the phosphorylation of endogenous PKM2, which was co-immunoprecipitated with JNK1 from the same lysates (IP:JNK1 and WB:PKM2). ( c ) In vitro JNK1 KA was performed by incubating recombinant activated JNK1 (rec. active JNK1) with His-PKM2 as substrate. [32P]-Rec. active JNK1 denotes autophosphorylation. In vitro pull-down assays were performed by IP and WBs after incubating purified rec. active JNK1 and His-PKM2 (IP:JNK1 and WB:PKM2). Coomassie staining shows the purity and size of the recombinant proteins. ( d ) Activated JNK1 was immunoprecipitated (IP:JNK1 and WB:p-JNK) from lysates of HEK293T cells expressing JNK1 constitutive active (JNK1 CA ) and assayed for kinase activity (KA) using recombinant His-PKM2 as substrate in the presence of [32P]-γ-ATP. [32P]-MKK7-JNK1α1 denotes the autophosphorylation of transfected JNK1 CA . ( e ) PKM2 activity was evaluated mixing recombinant His-PKM2 protein with different amounts of purified Rec. active JNK1 in the presence of PEP and ADP. The resultant formation of ATP serves as an internal catalyst for the in vitro JNK1-mediated phosphorylation/activation of PKM2 (top scheme). Data shown are mean±s.e.m. of three biological replicates. P values were calculated by one-way analysis of variance ( P <0.0001) followed by Bonferroni's multiple comparison tests. ( f ) WBs showing levels of phospho-PKM2(Tyr105) (p-PKM2(Tyr105)) in HCC cells expressing shPARP14 or control shNS. Total PKM2 serves as loading control. ( g ) Immunoprecipitation (IP) of PKM2 followed by WBs analyses detecting acetylated lysine (ac.-lys) in Hep3B cells expressing shPARP14 or control shNS. Total PKM2 and α-actinin were used as loading control. Ig, immunoglobulin.

Article Snippet: The full-length complementary DNAs of human PKM2 and PKM1 were obtained from pWZL-FLAG-PKM2 (Addgene plasmid 20585) and pET28-hPKM1 (Addgene plasmid 44241) , respectively, and then cloned between BamHI and XhoI sites of either pcDNA-HA- or pcDNA-FLAG-expressing vectors. pcDNA-HA-PKM2 mutant forms (S362A and T365A) were generated using the QuickChange XL site-directed mutagenesis kit (Stratagene).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Activity Assay, Recombinant, Phospho-proteomics, In Vitro, Purification, Staining, Activation Assay, Comparison, Control

Journal: Nature Communications

Article Title: PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation

doi: 10.1038/ncomms8882

Figure Lengend Snippet: ( a ) In vitro JNK1 KA performed by incubating recombinant activated JNK1 (rec. active JNK1) with His-tagged PKM2(WT), PKM2(S362A) or PKM2(T365A) as substrates. ( b ) PKM2 enzymatic activity was assessed incubating purified His-PKM2(WT) or His-PKM2(T365A) in the presence or absence of rec. active JNK1. ( c ) PKM2 enzymatic activity was evaluated in lysates of HEK293T cells transfected with JNK1 constitutive active (JNK1 CA ; 15 μg) or control empty vector (−) in combination with HA-PKM2(WT) or HA-PKM2(T365A) (15 μg). Levels of phosphorylated endogenous (endog.) JNK1 and JNK1 CA (MKK7-JNK1α1), and HA and PKM2 were visualized by WBs using appropriate antibodies. Total JNK1 serves as loading control. ( d ) Shown is the PK enzymatic activity, levels of reduced glutathione (GSH) and percentage of apoptotic cells in the sub-G1 (DNA content) population in PARP14-depleted HCC cells expressing empty vector (−), HA-PKM2(WT) or HA-PKM2(T365A). WBs showing the PARP14 knockdown efficiency and ectopic expression of HA-PKM2. ( b – d ) Data shown are mean±s.e.m. of three independent experiments. ( e ) Schematic illustration depicting metabolic changes in the presence (left) or absence (right) of PARP14 in HCC cells. The high levels of PARP14 expression in HCC cells halt JNK1-mediated phosphorylation of PKM2, which contribute to maintain low PKM2 activity required for the aerobic glycolysis. The low-active state of PKM2 promotes the Warburg effect and tumour cell survival by diverting glucose metabolites into alternative biosynthetic pathways and sustaining antioxidant responses . Depletion of PARP14, on the other hand, unleashes ‘active JNK1' to phosphorylate PKM2 enhancing its metabolic PK activity necessary for the conversion of glucose in pyruvate, thus lowering antioxidant responses and promoting apoptosis.

Article Snippet: The full-length complementary DNAs of human PKM2 and PKM1 were obtained from pWZL-FLAG-PKM2 (Addgene plasmid 20585) and pET28-hPKM1 (Addgene plasmid 44241) , respectively, and then cloned between BamHI and XhoI sites of either pcDNA-HA- or pcDNA-FLAG-expressing vectors. pcDNA-HA-PKM2 mutant forms (S362A and T365A) were generated using the QuickChange XL site-directed mutagenesis kit (Stratagene).

Techniques: In Vitro, Recombinant, Activity Assay, Purification, Transfection, Control, Plasmid Preparation, Expressing, Knockdown, Phospho-proteomics