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: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: ID-related LGI3 is uniquely expressed in oligodendrocytes (A) Domain organization of human LGI3. SP, signal peptide; LRR, leucine-rich repeat domain; EPTP, epitempin domain; N- and C-cap, N- and C-terminal cysteine caps in LRR. (B) Mapping of the ID variant on the modeled LGI3 structure. Left, Asp331 is mapped as red spheres. Right, close-up view of the Ca 2+ -binding site in the LGI3 EPTP β-propeller. Ca 2+ and water molecules are shown as gray and red spheres, respectively. Sticks, Ca 2+ -interacting residues; dotted lines, hydrogen bonds. (C) Shown are western blots (WBs) of LGIs-V5 in the conditioned medium (secreted) and the cell lysates. Bottom, quantification of the amounts of secreted LGI proteins. n = 3 experiments. Mann-Whitney U test. ∗ p < 0.05. Mean ± SD. (D) Fluorescence in situ hybridization (FISH) analysis for Lgi3 , Lgi1 , and Lgi2 expression in the mouse brain (P125). Red, Lgi3 , Lgi1 , or Lgi2 ; green, Mbp . White arrows, Mbp + / Lgi3 + cells; yellow arrows, Mbp – / Lgi3 + cells; white arrowheads, Mbp + / Lgi1 – or Mbp + / Lgi2 – cells; yellow arrowheads, Mbp – / Lgi1 + or Mbp – / Lgi2 + cells. Nuclear DNA was stained by DAPI (blue). CC, corpus callosum; Cb, cerebellum; WM, white matter; L5/6, cortical layers V and VI. Scale bars, 10 μm. (E) WB analysis of LGI family proteins in the lysates of the mouse brains and primary cultured cortical neurons. N-cadherin, a loading control. P, postnatal days; DIV, days in vitro . (F) WB analysis of Lgi3 LoxP/ΔE1 and Lgi3 LoxP/ΔE1 ; Mbp -Cre mouse brain lysates (P35). See also Figure S1 and Table S1 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Variant Assay, Binding Assay, Western Blot, MANN-WHITNEY, Fluorescence, In Situ Hybridization, Expressing, Staining, Cell Culture, Control, In Vitro

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 is clustered at the JXP of myelinated axons (A) Schematic image of FLAG-Hisx6 (FH)-tagged LGI3 protein. (B) Distributions of LGI3-FH (red) and LGI1 (green) in the mouse brain. (C) LGI3-FH is co-localized with MBP in the subcortical white matter (P166). Insets, wild-type mouse brain. (D) Biochemical subcellular fractionation of LGI3 in the mouse brain (P34). (E) LGI3-FH is not detected in the soma of oligodendrocytes in Lgi3 FH/FH mice (P183). CC-1, cytoplasmic oligodendrocyte marker protein. White arrowheads, LGI3-FH clusters; yellow arrowheads, somata of oligodendrocytes. (F and G) LGI3 is clustered at the JXP in the mouse brain and sciatic nerves. Sections are triple labeled by antibodies to (F) FH tag (LGI3-FH, red), Caspr1 (green, a paranode marker), and ankyrinG (blue, a node marker); (G) Hisx6 tag (LGI3-FH, green), Caspr2 (red, a JXP marker), and ankyrinG (blue, in the merged image) in the internal capsule. Arrows (in F, bottom) indicate LGI3-FH signals at the JXP. CC, corpus callosum; Str, striatum; Cb, cerebellum; WM, white matter; SN, sciatic nerves. P100 mouse tissue sections were used (B, F, and G). Scale bars, 1 mm (B), 250 μm (C), 10 μm (E and F, top), and 5 μm (F, bottom, and G). See also Figure S2 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Fractionation, Marker, Labeling

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 interacts with ADAM23 family, LGI family, and Kv1 channel proteins and co-clusters at the JXP in the brain (A) In vivo LGI3-associated protein complex purified from the Lgi3 FH/FH mouse brain (∼P100). Asterisks, specific bands co-purified with LGI3-FH. (B) LGI3-interacting protein-protein networks in the mouse brain. Thresholds for enrichment are shown by gray dashed lines. Light gray dots, proteins with subthreshold values; gray dots above thresholds, proteins in other categories. All protein data (997 proteins) for the plot are shown in Table S2 . n = 2 independent TAP experiments (with two shotgun MS per sample). (C) Shown are WBs of input and TAP eluates with the indicated antibodies. (D and E) Co-localization of LGI3 with ADAM23 family proteins and Kv1.2 channels at the JXP. (F) The endogenous LGI1 protein (endoLGI1) ( Lgi1 +/+ , left) and neuronally expressed LGI1-FH driven by Thy1 promoter in an Lgi1 KO background ( Lgi1 −/− ; Thy1 - Lgi1 -FH mouse) were detected at the JXP. (G) Localization of LGI2 at the JXP in the cerebellar white matter (Cb). P100 (D, E, and G) and P36 (F) mouse brain sections were used. Scale bars, 10 μm (D and E, left), 5 μm (F and G), and 1 μm (D and E, right). See also Figures S3 and and Tables S2 and .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: In Vivo, Purification

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 and its associated proteins form nanoscale subclusters at the JXP (A) Two-color (2C) STED imaging of Caspr1 and ADAM23 at the perinodal axon in corpus callosum (CC). (B) STED imaging of the large axons in the medulla. Dashed boxes are magnified in the middle two images (proximal and distal). The size of subclusters at the distal JXP is measured (bottom graphs). Red lines represent the mean value. FWHM, full width at half maximum. (C) 2C STED imaging of the juxtaparanodal proteins. P100 Lgi3 FH/FH mouse brain sections were used. Scale bars, 1 μm (A, left, and B, top) and 100 nm (A and B, magnified, and C). See also Figure S5 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Imaging

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: Loss of LGI3 specifically reduces ADAM23, ADAM22, and Kv1 channels in the myelin fraction without affecting myelination (A) Shown are representative WBs of the whole-brain lysates and subcellular fractions of wild-type ( +/+ ) and Lgi3 −/− mice (∼P100) (left). The graph shows protein levels of the Lgi3 −/− mouse brains relative to those of wild type (right). n = 5 mice per genotype. Mann-Whitney U test. ∗∗ p < 0.01. Mean ± SD. (B) Immunostaining for MBP in the neocortex and subcortical regions of the young (P10) and adult (P100) mouse brains. CC, corpus callosum; Str, striatum; Ctx, cortex. (C) Immunohistochemistry for CC-1 (mature oligodendrocyte marker, green) and Olig2 (expressed throughout the oligodendrocyte lineage, red) in the CC (P50). Nuclear DNA was stained by TO-PRO3 (blue). Bottom, quantification of the number of CC-1 + cells and Olig2 + cells. n = 3 mice per genotype. Two-tailed Student’s t test. NS, not significant. Mean ± SD. Scale bars, 100 μm (B) and 50 μm (C). See also Figure S6 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: MANN-WHITNEY, Immunostaining, Immunohistochemistry, Marker, Staining, Two Tailed Test

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: Loss of LGI3 disturbs ADAM23 and Kv1 channel clustering at the JXP (A and B) Immunohistochemistry for Kv1.1 and axonal domain marker proteins in the L5/6 (A) and CC (B) of the wild-type ( +/+ ) and Lgi3 −/− mice (left). Magnified images are shown at the bottom. The number and size of Caspr1 clusters and the number and length of juxtaparanodal Kv1.1 clusters are quantified (right graphs). (C and D) The number of ADAM23 clusters at the JXP is significantly reduced in the L5/6 and CC of Lgi3 −/− mice (C), while that of ADAM11 clusters is not affected (D). (E) Representative STED images of juxtaparanodal Kv1.1 clustering in the L5/6 of wild-type ( +/+ ) and Lgi3 −/− mice. (F) Schematic illustration of stereotaxic injection of AAV for Adam23 family gene KO by the CRISPR-Cas9 system. AAV-DJ-expressing sgRNA for Adam23 or Adam22 and a reporter mCherry were injected into the primary motor cortex of Rosa26 Cas9/Cas9 knockin mice (∼P40). Juxtaparanodal cluster formation on the mCherry + axons in the contralateral cortex (blue square) was evaluated at 8 weeks after injection. (G) Representative images of the juxtaparanodal proteins (grayscale or blue) on the mCherry + (red) axons are shown. The number of juxtaparanodal protein clusters (ratio to Caspr1 clusters, green) was significantly reduced in the Adam23 KO axons. (A–E) Wild-type mice, P100–P119; Lgi3 −/− mice, P100. (A–D) n = 3 mice per genotype. Mann-Whitney U test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001. Mean ± SD. (G) n = 4 experiments, one-way ANOVA post hoc Tukey’s test. NS, not significant. ∗∗ p < 0.01. mean ± SD. Scale bars, 10 μm (A and B, top), 5 μm (A and B, bottom, C, and D), 1 μm (E), and 10 μm (G). See also Figure S7 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Immunohistochemistry, Marker, Injection, CRISPR, Expressing, Knock-In, MANN-WHITNEY

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 regulates short-term synaptic plasticity through juxtaparanodal Kv1 channels (A) Schematic electrophysiological experiments. Blue and green circles represent light illumination to the white matter and to the center of recording L5a pyramidal cell (PC) soma, respectively. (B) Representative averaged current trace of L5a PC in response to repetitive stimulations by light illumination (1 ms duration, 20 Hz, light blue bars) to the white matter axons of ChR2 + L2/3 callosal neurons. (C and D) EPSC amplitude ratio (second to first and third to first responses, respectively) of L5a PCs in the wild-type ( +/+ ) or Lgi3 −/− mouse brain slices evoked by white matter (C) or soma stimulation (D). Wild-type, n = 13 and 12; Lgi3 −/− , n = 16 and 15 (white matter and soma stimulation, respectively). Welch’s t test. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001, and ∗∗∗∗ p < 0.00001. Mean ± SD. (E and F) Blockade of Kv1 channels suppresses STD inductions in wild-type but not in Lgi3 −/− mice. Ratio of EPSC evoked by white matter stimulation before and during bath application of (E) 100 μM 4-AP and (F) 100 nM DTX-I. Wilcoxon signed-rank test. NS, not significant. ∗ p < 0.05. (G and H) Presynaptic terminals on recorded L5a PCs were directly stimulated by soma stimulation in the presence of 1 μM TTX before and during bath application of 100 μM 4-AP (G) or 100 nM DTX-I (H). Wilcoxon signed-rank test. NS, not significant. (I) LGI3 is exclusively localized in non-presynaptic regions of the mouse cortex in contrast to LGI1. Dashed white square area is magnified (bottom). Manders co-localization coefficient of vGlut1/2 with LGI3 or LGI1 is shown in the graph. Scale bars, 10 μm (top) and 5 μm (magnified, bottom). n = 3 experiments. Mean ± SD. (J) Presynaptic Kv1 channels are not altered in Lgi3 −/− mice. The intensity of the Kv1.2 is quantified (graph). n = 6 experiments. Two-tailed Mann-Whitney U test. NS, not significant. Mean ± SD. Scale bars, 10 μm. (K and L) Loose-patch single-axon recordings in the CC. (K) Inset shows the schematic loose-patch single-axon recording. S, stimulating electrode. (L) Wilcoxon signed-rank test. NS, not significant. ∗ p < 0.05. Red lines represent the mean value (E–H and L). See also Tables S4 and .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet:

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Recombinant, Virus, Sequencing, Modification, RNAscope, Multiplex Assay, Positive Control, Negative Control, Mass Spectrometry, Knock-In, Knock-Out, Transgenic Assay, Control, Software

Journal: PLoS ONE

Article Title: Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2

doi: 10.1371/journal.pone.0191419

Figure Lengend Snippet: (A) Recombinant human PKM1 enzyme was isolated and in vitro PKM1 enzyme activity was measured using a continuous assay coupled to lactate dehydrogenase. Different concentrations of lapachol (vehicle, 5 μM and 10 μM) did not show an inhibition in the activity of recombinant PKM1 invitro. (B) lapachol inhibited the activity of recombinant PKM2 in a dose dependent manner (n = 3/condition) **** P<0.0001 (C) Lapachol inhibited the pyruvate kinase activity invivo in the melanoma cell line MEL103 in a dose dependent manner (n = 3/condition)****P<0.0001.

Article Snippet: Human PKM1 (#44241) and PKM2 (#44242) in pET28a vector were purchased from addgene expressed as an N-terminal His 6 tag fusion protein. pET28a-PKM1 and pET28a-PKM2 was transformed into BL2 (DE3) pLysS cells and grown to an absorbance of 0.8 at 600 nm, then induced with 0.5 mM IPTG for 7 h at room temperature.

Techniques: Recombinant, Isolation, In Vitro, Activity Assay, Inhibition