Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a, Enrichment of GO terms among CDK6-interactors identified in all T-ALL cell lines. p-values, Benjamini-Hochberg-test. b, In vitro kinase reactions using immunoprecipitated endogenous CDK6 and recombinant PFKP or PKM2 ±palbociclib (PALBO). 32P-PFKP/PKM2 denotes phosphorylated proteins, IB, immunoblotting. c, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 2e). d, PFKP and PKM2 activity in cells transfected with empty vector (Vec), D3/CDK6, or kinase-dead CDK6 (D3/CDK6-KD). e, PFKP and PKM2 activity after palbociclib-treatment. Data are mean ±s.d. *P<0.05; **P<0.01 (two-tailed t-test). b,c, representative experiments, out of 2 independent experiments (b), or 3 independent experiments (c, error bars from technical replicates). d,e, n=3 biological replicates. See Supplementary Fig. 1 for gel source data.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: In Vitro, Immunoprecipitation, Recombinant, Western Blot, Phospho-proteomics, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: Flow of glucose-derived carbon into PPP (a), and serine pathway (b) following D3-CDK6 inhibition in T-ALL KOPTK1 cells expressing wild-type PFKP and PKM2 (KOPTK1-WT), or PFKP-S679E and PKM2-S37E mutants (KOPTK1-EE). Levels of NADPH (c), GSH (d), ROS (e) in T-ALL cell lines upon palbociclib-treatment. f, Apoptosis of cells treated with palbociclib and NAC. g, Apoptosis in KOPTK1-WT and KOPTK1-EE cells upon palbociclib-treatment, or following knockdown of CDK6 (h). i,j, In vivo apoptosis of T-ALL cells (in peripheral blood and bone marrow, gated on human CD45+cells) in mice xenografted with KOPTK1-WT or KOPTK1-EE cells. Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, n=4, c-h, n=3, i,j, n=5 biological replicates. See Source data for Fig. 2 for T-ALL xenograft experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Derivative Assay, Inhibition, Expressing, Knockdown, In Vivo, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a,b, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 9i), PFKP and PKM2 activity (c,d), levels of NADPH (e), GSH (f), ROS (g) in palbociclib-treated D3/CDK6-high cells. h, Apoptosis of D3/CDK6-high EBC1 cells expressing wild-type PFKP and PKM2 (EBC1-WT), or PFKP-S679E and PKM2-S37E mutants (EBC1-EE). Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, representative experiments (out of 3 independent experiments, error bars from technical replicates), c-h, n=3 biological replicates.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: PFKP and PKM2 phosphorylation (a,b, from Extended Data Fig. 10i, j), PFKP and PKM2 activity (c,d), GSH (e) and ROS (f) levels in D3/CDK6-high (regressing) and D3/CDK6-low (non-regressing) tumors from ribociclib-treated mice. Data are mean ±s.d. P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). n=3 biological replicates. See Source data for Fig. 5 for PDX drug treatment experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test

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 Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Expression of PKM2 determines glycometabolism rate and sensitivity to oxaliplatin in SW480, OR-SW480, HT29, and OR-HT29 cells (A) Expression of PKM2 in SW480, OR-SW480, HT29, and OR-HT29 was evaluated by quantitative real-time PCR and western blot analysis. ∗p < 0.05. (B) Transfection efficiency of PKM2 siRNA (50 pmol/mL) and PKM2 plasmid (2 μg/mL) in SW480, OR-SW480, HT29, and OR-HT29 cells. (C) Glucose, lactate, and ATP assays were performed to evaluate the glycometabolism of SW480 and HT29 cells after treatment with oxaliplatin (1 μM) and PKM2 plasmid (2 μg/mL). ∗p < 0.05 versus empty plasmid group. (D) Glucose, lactate, and ATP assays were performed to evaluate the glycometabolism of OR-SW480 and OR-HT29 cells after treatment with oxaliplatin (10 μM) and PKM2 siRNA (50 pmol/mL). ∗p < 0.05 versus NCO group. # p < 0.05 versus Oxaliplatin + NCO group. (E) Effect of PKM2 plasmid (2 μg/mL) on inducing the oxaliplatin resistance in SW480 and HT29. ∗p < 0.05 versus Oxaliplatin + empty plasmid group. (F) Effect of PKM2 siRNA (50 pmol/mL) on reversing the oxaliplatin resistance in OR-SW480 and OR-HT29. ∗p < 0.05 versus oxaliplatin + NCO group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Scutellarin decreases the oxaliplatin resistance through inhibition of PKM2 in OR-SW480 and OR-HT29 cells (A) Effect of scutellarin (2 μM), oxaliplatin (10 μM), and PKM2 plasmid (2 μg/mL) on changing the expression of PKM2 in OR-SW480 and OR-HT29 cells. (B) Effect of PKM2 plasmid (2 μg/mL) on protecting the OR-SW480 and OR-HT29 cells from the cytotoxicity of co-treatment with oxaliplatin (10 μM) and scutellarin (2 μM). ∗p < 0.05 versus oxaliplatin + empty plasmid group. # p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group. (C) PKM2 plasmid (2 μg/mL) increased the glycometabolism rate in scutellarin-treated (2 μM) OR-SW480 and OR-HT29 cells. ∗p < 0.05 versus empty plasmid group. # p < 0.05 versus oxaliplatin + empty plasmid group. & p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Inhibition, Plasmid Preparation, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Promotion of scutellarin on oxaliplatin-induced mitochondrial apoptosis pathway (A) Mitochondrial membrane potential (Δϕ) of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL) was measured by flow cytometry. (B) Release of cytochrome c and AIF was evaluated by western blot analysis after removal of mitochondria from cytosol of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). (C) Western blot analysis was performed to detect the cleavage of caspase-9 and caspase-3 in OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). (D) Flow cytometry analysis was performed to detect the apoptotic rate of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). ∗p < 0.05 versus empty plasmid group. # p < 0.05 versus oxaliplatin + empty plasmid group. & p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Flow Cytometry, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Scutellarin sensitizes oxaliplatin-resistant CRC cells to oxaliplatin treatment in vivo (A) Tumor growth of mice bearing OR-SW480 cells after treatment with oxaliplatin (10 mg/kg) and scutellarin (10 mg/kg) twice a week. (B) Resected tumors from mice in each group. (C) Western blot assay was performed to detect the cleavage of caspase-9 and caspase-3 in the resected tumors. (D) Western blot assay was performed to analyze the expression of PKM2 in the resected tumors. (E) Production of ATP in the purified tumor cells in each group. ∗p < 0.05 versus control group. # p < 0.05 versus oxaliplatin group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: In Vivo, Western Blot, Expressing, Purification

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Schema of the predicted mechanisms implicated in OR-SW480 cells response to oxaliplatin Scutellarin inhibits PKM2 expression and thus reduces the glycometabolism rate and the production of ATP. The lower level of ATP facilitates the oxaliplatin-induced mitochondrial dysfunction, as determined by a decrease in Δϕ. As a result, cytochrome c and AIF are released from the mitochondria into the cytosol. Subsequently, these apoptotic inducers activate the effector caspases and cause the final occurrence of apoptosis.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing

Journal: Angiogenesis

Article Title: KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

doi: 10.1007/s10456-015-9475-4

Figure Lengend Snippet: HIF regulates the Warburg effect in KSHV-infected cells. a HIF1α and GFP expression and DAPI staining in HUVEC and KSHV cells. b HUVEC and KSHV cells were transfected with pGL-VEGF/K and pCEFL Renilla luciferase and exposed to vehicle (−) or 100 nM digoxin (+) for 24 h. Luciferase activity and Renilla activity (for normalization) were determined as described in “Materials and methods” section. c, d Real-time PCR of HIF angiogenic genes (ANGPT2, ANGPTL4, VEGF) (c) and HIF metabolic genes (PKM2, HK2, GLUT1, BNIP3, PDK1) (d) in HUVEC and KSHV cells. Cells were treated with vehicle (−) or 100 nM digoxin (+) for 24 h. e Extracellular lactate concentration and ratio of mitochondrial DNA of cells exposed to vehicle (−) or 100 nM digoxin (+), or transfected with no siRNA (No si) scrambled siRNA (Scr si) or HIF1β siRNA (HIF1β si). Western blots show levels of HIF1α, HIF2α, or HIF1β in the corresponding treated/untreated or transfected/untransfected cells

Article Snippet: Expression vectors (pcDNA3.1-V5-His) encoding PKM1, PKM2, or PKM2 K270M or (pCEFL myc) HIF1αΔODD have been described elsewhere [ 21 , 41 ]. siRNAs oligos (HIF1β, PKM2) were purchased from Qiagen.

Techniques: Infection, Expressing, Staining, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Western Blot

Journal: Angiogenesis

Article Title: KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

doi: 10.1007/s10456-015-9475-4

Figure Lengend Snippet: PKM2 regulates KS cell metabolism. a PKM1, PKM2, and HIF1α protein levels in HUVEC, IN-KSHV, and KSHV cells and PKM2, HIF1α, and HIF2α protein levels in HUVEC and KSHV cells, treated with vehicle (−) or 100 nM digoxin (+) for 24 h. b H&E staining and PKM2 immunohistochemical staining of human AIDS-related KS biopsy. c Effect of overexpression of (pcDNA3.1-V5-His) PKM1 or PKM2 or (pCEFL myc HIF1αΔODD) HIF1α in HUVEC on lactate production, oxygen consumption, mitochondrial DNA content, and cell proliferation (calculated as described in “Materials and methods” section). Hypoxia treatment (1 % O2) was used as a control. d Effect of the expression of no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si), in HUVEC or KSHV cells on lactate production, oxygen consumption, mitochondrial DNA content, and cell proliferation. e Corresponding levels of PKM2 upon transfection of no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si), in HUVEC or KSHV cells

Article Snippet: Expression vectors (pcDNA3.1-V5-His) encoding PKM1, PKM2, or PKM2 K270M or (pCEFL myc) HIF1αΔODD have been described elsewhere [ 21 , 41 ]. siRNAs oligos (HIF1β, PKM2) were purchased from Qiagen.

Techniques: Staining, Immunohistochemical staining, Over Expression, Expressing, Transfection

Journal: Angiogenesis

Article Title: KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

doi: 10.1007/s10456-015-9475-4

Figure Lengend Snippet: VEGF expression is regulated by PKM2 in KSHV-infected cells. a Immunofluorescent staining of PKM2 and HIF1α in IN-KSHV and KSHV cells. b Real-time PCR analysis of HIF downstream genes (BNIP3, GLUT1, HK2, LDHA, VEGF) in KSHV cells expressing (control) scrambled siRNA or PKM2 siRNA. c, d HUVEC (c) or KSHV (d) cells were transfected with increasing doses of PKM1, PKM2, and PKM2 K270M expression vector (pcDNA3.1-V5-His) (c) or increasing doses of PKM2 siRNA (d), along with pGL-VEGF/K and pCEFL Renilla luciferase. Luciferase activity and Renilla activity (for normalization) were determined as described in “Materials and methods” section. e Levels of VEGF protein expression and VEGF secretion of HUVEC and KSHV expressing no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si)

Article Snippet: Expression vectors (pcDNA3.1-V5-His) encoding PKM1, PKM2, or PKM2 K270M or (pCEFL myc) HIF1αΔODD have been described elsewhere [ 21 , 41 ]. siRNAs oligos (HIF1β, PKM2) were purchased from Qiagen.

Techniques: Expressing, Infection, Staining, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Angiogenesis

Article Title: KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

doi: 10.1007/s10456-015-9475-4

Figure Lengend Snippet: Inhibition of PKM2 expression blocks KSHV-infected cell angiogenicity. a, b Cell migration (modified Boyden chamber assay) assay (a) and tube formation assay (matrigel assay) (b) in HUVEC or KSHV cells expressing no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si). c Conditioned media from HUVEC or KSHV cells expressing no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si) was used to induce blood vessel development in BME-filled angioreactors implanted in nude mice (DIVAA assay)

Article Snippet: Expression vectors (pcDNA3.1-V5-His) encoding PKM1, PKM2, or PKM2 K270M or (pCEFL myc) HIF1αΔODD have been described elsewhere [ 21 , 41 ]. siRNAs oligos (HIF1β, PKM2) were purchased from Qiagen.

Techniques: Inhibition, Expressing, Infection, Migration, Modification, Boyden Chamber Assay, Tube Formation Assay, Matrigel Assay

Journal: Angiogenesis

Article Title: KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

doi: 10.1007/s10456-015-9475-4

Figure Lengend Snippet: Inhibition of PKM2 inhibits vGPCR oncogenesis. a PKM2 analysis upon treatment of HUVEC cells with control or vGPCR conditional media (CM) over time. b PKM2 analysis upon treatment of HMEC or HUVEC cells with control or vGPCR conditional media (CM). Cells were treated with vehicle (−), rapamycin (50 nM, 24 h, R), digoxin (100 nM, 24 h, D). Levels of RhoA serve as negative control. c Immunohistochemistry of PKM2 in vGPCR tumor allograft [30]. d, f Effect of PKM2 knockdown (no siRNA (No si), scrambled siRNA (Scr si) or PKM2 siRNA (PKM2 si) on VEGF expression and secretion (d), endothelial cell differentiation (tube formation assay) (e), and in vivo blood vessel development (DIVAA assay) (f). g PKM2 knockdown blocks vGPCR-induced allograft growth in nude mice. Mice were injected in the flank with (1:10) mixed populations of SVEC stably expressing vGPCR and cells expressing no shRNA (No sh), scrambled shRNA (Scr sh) or PKM2 shRNA (PKM2 sh). Percentage of tumor growth is shown

Article Snippet: Expression vectors (pcDNA3.1-V5-His) encoding PKM1, PKM2, or PKM2 K270M or (pCEFL myc) HIF1αΔODD have been described elsewhere [ 21 , 41 ]. siRNAs oligos (HIF1β, PKM2) were purchased from Qiagen.

Techniques: Inhibition, Negative Control, Immunohistochemistry, Expressing, Cell Differentiation, Tube Formation Assay, In Vivo, Injection, Stable Transfection, shRNA