Journal: bioRxiv

Article Title: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma

doi: 10.1101/2021.05.09.443334

Figure Lengend Snippet: A. A schematic showing the mouse Pkm locus, construct targeting Pkm1 -specific exon 9, and the resulting targeted, floxed, and deleted Pkm1 alleles. The KpnI restriction enzyme sites used for Southern blot analysis are marked with “K,” and the new KpnI site introduced by the targeting vector is marked with “K*”. The location of the 5’ probe used for southern blot analysis is also indicated, as are the locations of the genotyping primers (green arrows). B. Southern blot analysis of KpnI-digested genomic DNA from Pkm1 +/+ (+/+), Pkm1 +/fl (f/+), and Pkm1 fl/fl (f/f) mice using the 5’ probe shown in (A). Digestion of genomic DNA harboring the wild-type allele (+) yields an 8.3 kb fragment, while DNA harboring the floxed allele (f) yields a ∼5.0kb fragment. C. Pkm1 mRNA levels in Pkm1 +/+ and Pkm1 -/- anterior prostate tissue as determined by quantitative RT-PCR. Mean+/-SD is shown (n=5). The difference in expression between genotypes is significant (***p<0.001 by Student’s t-test). D. Western blot analysis of Pkm1 , Pkm2 , and vinculin expression in various tissues including the ventral, anterior, and dorsolateral (DLP) prostate lobes that were harvested from wild type and Pkm2 pc-/- mice as indicated.

Article Snippet: Western blots were performed using primary antibodies against Pkm1 (Sigma SAB4200094), Pkm2 (Cell Signaling Technology #4053), Pkm (Cell Signaling Technology #3190; Abcam ab6191), and Vinculin (Sigma, V4505, clone VIN-11-5).

Techniques: Construct, Southern Blot, Plasmid Preparation, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma

doi: 10.1101/2021.05.09.443334

Figure Lengend Snippet: A. Southern blot screening of embryonic stem cell clones for homologous recombination of the Pkm1 targeting construct using the 5’ probe indicated in . Insertion of a novel KpnI site results in a new ∼5.0 kb fragment from the targeted locus following digestion of genomic DNA. Two clones with successful integration are marked with an arrowhead. B. PCR genotyping of genomic DNA from Pkm1 +/+ (+/+), Pkm1 fl/+ (f/+), and Pkm1 fl/fl (f/f) mice as indicated. Genotyping primers anneal at sites indicated in to produce amplicons of 509 bp from the Pkm wild-type allele ( Pkm + ) and 577 bp from the conditional Pkm allele with flanking LoxP sites ( Pkm1 fl ).

Article Snippet: Western blots were performed using primary antibodies against Pkm1 (Sigma SAB4200094), Pkm2 (Cell Signaling Technology #4053), Pkm (Cell Signaling Technology #3190; Abcam ab6191), and Vinculin (Sigma, V4505, clone VIN-11-5).

Techniques: Southern Blot, Clone Assay, Homologous Recombination, Construct

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 promotes pyruvate kinase activity and suppresses serine synthesis. A, Carm1 knockout (−/−) MEFs exhibit lower PK activity than WT MEFs. In vitro PK assays were performed using total cell lysates of WT and Carm1−/− MEFs. B, re-expression of GFP-CARM1 in Carm1−/− MEFs restores PK activity. The rescued expression of CARM1 was confirmed by Western blot analysis (left panel). PK activity assays (right panel) were performed using total cell lysates from untransfected (−) and vector- or G-CARM1–transfected Carm1−/− MEFs. Actin was used as a loading control. C, expression of PKM1 and PKM2 was examined in WT and Carm1−/− MEFs by Western blot analysis. D, re-expression of FLAG-PKM1 (F-PKM1) in Carm1−/− MEFs restores PK activity. The expression of PKM1 was confirmed by Western blot analysis (left panel). PK activity assays (right panel) were performed using total cell lysates from the indicated cells. E, overexpression of PKM1 in Carm1−/− MEFs suppresses de novo serine synthesis. The level of 13C-labeled serine in the indicated cells was measured using GC-MS. Data represent mean ± S.E., n = 3.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Activity Assay, Knock-Out, In Vitro, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Over Expression, Labeling, Gas Chromatography-Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 methylates PKM2 at Arg-445 and Arg-447 sites. A, PKM2 is methylated by CARM1. In vitro methylation assays were performed by incubating GST-tagged recombinant PRMTs (PRMT1, PRMT3, CARM1, Myc-PRMT5, and PRMT6) with purified His-tagged PKM2 proteins. A, B, and E, arrows indicate PKM2 methylation, and solid dots indicate PRMT automethylation. Membranes were stained with Ponceau S to ensure equal protein loading. B, recombinant human PKM1 (hPKM1) and PKM2 (hPKM2), mouse PKM2 (mPKM2), and tetramerization-deficient mutant mPKM2 (R399E) were subjected to in vitro methylation assays with CARM1. C, LC-MS/MS was performed to identify the arginine methylation sites of in vitro methylated His–mPKM2. Arginines 445 and 447 were found to be both mono- and dimethylated. D, PKM2 arginine methylation sites, Arg-445 and Arg-447, share sequence consensus with several known CARM1 substrates, including poly(A)-binding protein 1 (PABP1), histone H3R17, and cAMP-response element-binding protein-binding protein (CBP). E, in vitro methylation assays were performed using WT and R445K/R447K mutant PKM2, confirming that Arg-445 and Arg-447 are the major sites of methylation by CARM1.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, In Vitro, Recombinant, Purification, Staining, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Sequencing, Binding Assay, Protein Binding

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: Arginine methylation PKM2 promotes its PK activity in vitro. A, arginine-methylated PKM2 exhibits significantly greater PK activity than unmethylated PKM2. Recombinant PKM2 was subjected to in vitro methylation by either WT or catalytic-deficient CARM1 (R169A) in the presence or absence of SAM. The methylation products were dialyzed and subjected to in vitro PK activity assays (left panel). In vitro methylation assays were performed to confirm that CARM1 (R169A) is catalytically deficient in methylating PKM2 (right panel). *, p < 0.05. B, CARM1 promotes PKM2 PK activity through arginine methylation. Recombinant WT and methylation-defective PKM2 (R445/447K) were subjected to in vitro methylation by CARM1. The reaction products were dialyzed and subjected to in vitro PK activity assays. NS, not significant. C, structural analysis of PKM2 reveals that methylation of Arg-447 (R447me2a) potentiates intramolecular interactions with distal amino acids, Leu-392 and Phe-421. D, mutations of amino acids involved in the intramolecular interactions of PKM2 abolish methylation-mediated regulation of PKM2 activity. E, recombinant WT, methylation-deficient (R445/447K), and intramolecular interaction-deficient (L392A, F421A, and L392A/F421A) PKM2 were subjected to in vitro methylation by CARM1. The reaction products were dialyzed and subjected to in vitro PK activity assays.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, Activity Assay, In Vitro, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: Arginine methylation enhances PKM2 tetramerization. A, arginine methylation-deficient PKM2 (R445/447K) expressed in mammalian cells is less active than the WT enzyme. FLAG-tagged WT and R445K/R447K mutant PKM2 were expressed and purified from MCF7 cells (left panel). Eluted proteins were subjected to in vitro PK activity assays (right panel). B, loss of arginine methylation reduces PKM2 intermolecular interactions. Co-immunoprecipitation assays were performed to examine the interactions of endogenous PKM2 with FLAG-tagged WT or arginine methylation-deficient (R445/447K) PKM2 in MCF7 cells. * indicates the IgG heavy chain. Black arrow indicates transfected FLAG-tagged constructs, and white arrow indicates the endogenous PKM2. C, arginine methylation promotes PKM2 tetramerization. MCF7 cells were transfected with FLAG-tagged WT and arginine methylation-deficient PKM2 (R445/447K). Total cell lysates from transfected cells were separated by gel filtration, followed by Western blot analysis using an anti-FLAG antibody to determine the proportion of mono-, di-, and tetramers (indicated with open circles above the gels). D, reduced PKM2 tetramerization in Carm1−/− MEF cells. Total cell lysates from WT and Carm1 knockout (−/−) MEFs were separated by gel filtration, followed by Western blot analysis using an anti-PKM2 antibody.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, Mutagenesis, Purification, In Vitro, Activity Assay, Immunoprecipitation, Transfection, Construct, Filtration, Western Blot, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 knockout cells are more resistant to serine deprivation than WT cells, likely due to reduced PK activity. A, CARM1 knockout (−/−) MEFs are more resistant to serine deprivation. The proliferation of WT and Carm1−/− MEFs in complete (left panel) and serine-free (right panel) medium was assayed over a 4-day period. B, WT and CARM1−/− MEFs respond similarly to the PKM2 small molecule activator TEPP-46. To activate PK activity, WT and Carm1−/− MEFs were either untreated or treated with TEPP-46 (25 μm) for 24 h. The total cell lysates were subjected to in vitro PK activity assays. C, activating PK activity using PKM2 activator TEPP-46 abolishes the survival advantage of CARM1−/− MEFs in serine-free medium. WT and Carm1−/− MEFs were treated with DMSO or TEPP-46 (25 μm) and subjected to culture in serine-free medium over a 4-day period. The cell proliferation was monitored as described in A.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Knock-Out, Activity Assay, In Vitro

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Proportion of PKM2 expression in cells of different histologic types of benign and malignant ovarian tumor. (B) Representative differential PKM2 expression in benign and malignant groups of epithelial ovarian tumor. * p < 0.001. (C) Proportion of p53 and PKM2 expression in cells of different histologic models of ovarian cancer. (D) Spearman's rho showed moderate correlations between PKM2 and p53 expression. PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: Clinicopathological features.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Kaplan–Meier analysis of the probability of progression-free survival ( p = 0.01) (left) and overall survival ( p = 0.057) (right) in patients with ovarian cancer, stratified according to PKM2 expression ( n = 88). (B) Expression of PKM2 in different ovarian cancer cell lines. (C and D) Time-dependent inhibition of glucose consumption (** p< 0.01,*** p< 0.001) and lactate production in CP70 and SKOV3 cells following shikonin treatment. PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing, Inhibition

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for progression-free and overall survival in patients with ovarian cancer.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) The ECAR according to Seahorse analysis decreased following shikonin treatment in SKOV3 and CP70 cells (* p< 0.05). (B) PKM2 inhibition induced a shift in OCR/ECAR in SKOV3 cells. (C) Dose-dependent inhibition of cancer cell growth in CP70 and SKOV3 cells following shikonin treatment. (D) Migration assay involving CP70 and SKOV3 cells following shikonin treatment. ECAR, extracellular acidification rate; OCR, oxygen-consumption rate; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Inhibition, Migration

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) Treatment with shikonin did not cause differences in body weight loss between the treatment and control groups. (B) Representative micro-PET images of xenograft mice in the shikonin-treated group as compared with the control group. (C) Shikonin treatment inhibited tumor formation in vivo following injection of SKOV3 ovarian cancer cells. PET, positron emission tomography; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Control, Micro-PET, In Vivo, Injection, Positron Emission Tomography

Journal: PLoS ONE

Article Title: Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer

doi: 10.1371/journal.pone.0182166

Figure Lengend Snippet: (A) H&E and IHC stains of mouse tumors following shikonin treatment. (B) Shikonin treatment did not cause apparent pathologic abnormalities in the brain, kidney, liver, or heart according to H&E staining. H&E, hematoxylin and eosin; IHC, immunohistochemistry; PKM2, Pyruvate kinase M2.

Article Snippet: After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a rabbit polyclonal anti-human PKM2 antibody (Aviva Systems Biology, San Diego, CA, USA) diluted 1:100 in PBS, and bound antibodies were detected using a streptavidin-biotin-peroxidase system (Dako, Glostrup, Denmark) and 3,3 ́-diaminobenzidine substrate chromogen solution (Dako).

Techniques: Staining, Immunohistochemistry

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: International Journal of Molecular Sciences

Article Title: Creatine Kinase Blockade Disrupts Energy Metabolism and Redox Homeostasis to Suppress Osteosarcoma Progression

doi: 10.3390/ijms262311555

Figure Lengend Snippet: Effects of CK KD on glycolysis and metastasis. ( A ) Expression of glycolysis-related proteins (PFK2, PKM2, Akt, AMPK) measured by ELISA ( B ) Lactate production. ( C ) Circulating tumor cells at 12 h post-tail vein injection. Images were taken with a phase-contrast microscope. Each experimental group consisted of 10 mice. Scale bar, 100 μm. ( D ) Pulmonary colonization of fluorescently labeled OS cells. * p < 0.05 vs. siC. Data represent mean ± SD of 10 mice. Statistical differences were calculated using ordinary ANOVA with Bonferroni correction. ANOVA, analysis of variance; KD, knockdown; si, small interfering RNA; siC, control siRNA; siCK, siCKB + siMTCK1; PKM2, pyruvate kinase M2; PFK2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2; AMPK, 5′-AMP-activated protein kinase; FI, fluorescent intensity.

Article Snippet: human PKM2 , NBP3-18036 , Novus Biologicals, Centennial, CO, USA , .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Injection, Microscopy, Labeling, Knockdown, Small Interfering RNA, Control