Fig 1: LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured podocytes. Representative immunoblots of pPKM2Y105, pPKM2S37, PKM2, PKM1, PKM1/2, nephrin, and ß-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2Y105, pPKM2S37, PKM2, PKM1, PKM1/2, nephrin, and ß-Actin in cultured murine E11 podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to ß-Actin from three independent experiments. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells
Fig 2: JNK1 interacts with and activates PKM2 by phosphorylation.(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]-?-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 (JNK1CA) and assayed for kinase activity (KA) using recombinant His-PKM2 as substrate in the presence of [32P]-?-ATP. [32P]-MKK7-JNK1a1 denotes the autophosphorylation of transfected JNK1CA. (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 a-actinin were used as loading control. Ig, immunoglobulin.
Fig 3: (A) MCF7IGF1R-ve/IR-A cells with or without DDR1 silencing were analyzed by Western immunoblotting for DDR1 expression using polyclonal antibodies against the C-terminus of DDR1, as indicated. ß-actin antibody was used as control for protein loading. A representative blot of three independent experiments is shown. (B) ATP production rate in MCF7IGF1R-ve/IR-A cells silenced for DDR1 and in control cells treated with scramble siRNAs, upon stimulation with 100 nM IGF2 for 48 h. Glycolytic ATP (red columns—glycoATP) and mitochondrial ATP (blue columns—mitoATP) production rates were evaluated according to the manufacturer’s instructions as described above (Agilent ATP test). The presented histogram (left panel) and the energetic map (right panel) show the mean and range from three independent experiments. (C) MCF7DDR1-ve cells were analyzed by Western immunoblot for DDR1 expression using polyclonal antibodies against the C-terminus of DDR1, as indicated. A positive control is also shown. ß-actin antibody was used as control for protein loading. A representative blot of three independent experiments is shown. (D) ATP production rate in MCF7DDR1-ve silenced for DDR1 and in control cells treated with scramble siRNAs. The presented histogram (left panel) and the energetic map (right panel) show the mean and range from three independent experiments. (E) MCF7DDR1-ve cells and parental MCF7Cas9 control cells grown in complete medium were treated with siDDR1 or scramble siRNAs and analyzed for mRNA expression of transporters (MCT1, MCT4), glycolysis related enzymes (PKM2, EK2), and mitochondrial markers (PGC1ß, MFN1, PNC1, TFAM, NRF1, NRF-2a, and CYTOB). Normalization was performed using human GAPDH as a housekeeping control gene. Values are expressed as means ± SEM of three independent experiments. (F–G) MCF7DDR1-ve and MCF7Cas9 cells silenced or not for DDR1 were analyzed by SDS-PAGE and immunoblot for the indicated proteins. A representative blot of three independent experiments is shown. (ns—not significant; *, p < 0.05; **, p < 0.01; Student’s t-test).
Fig 4: PARP14 inhibits PKM2 activity to promote the Warburg effect.(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.
Fig 5: Activators promote PKM2 tetramer formation and prevent inhibition by pTyr signaling(a) Sucrose gradient ultracentrifugation profiles of purified recombinant PKM2 and effects of FBP and TEPP-46 on PKM2 subunit stoichiometry. Recombinant PKM2 was transiently exposed to FBP prior to addition of TEPP-46. After centrifugation, fractions were collected, analyzed by SDS-PAGE and stained with Coomassie Blue. Relative protein amounts were calculated by band densitometry on a LiCOR Odyssey infrared imaging system. (b) A549 cells were treated with 100 µM pervanadate for 10 min. in the presence or absence of TEPP-46, lysed hypotonically, and were analyzed by size exclusion chromatography. Chromatographic fractions were then subjected to western blotting with a pyruvate kinase antibody to assess the stoichiometry of PKM2 subunit association under these conditions. Uncropped blots are shown in Supplementary Fig. 10. (c) Pyruvate kinase activity assays in A549 cells treated with pervanadate as in (b) in the presence of DMSO, 1 µM TEPP-46 or 1 µM DASA-58 (N=3, p=0.0044 by 2-way ANOVA).
Supplier Page from MilliporeSigma for Anti-PKM2 (isoform M1) antibody produced in rabbit