mt4 vector  (Addgene inc)

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: PKM2 is a novel binding partner of PSAT1. ( A ) Silver stain of GST-PSAT1-purified proteins from A549 whole-cell lysates. * Denotes residual GST-PSAT1 from column purification; ← denotes gel slice encompassing PKM. ( B ) Primary amino acid sequence of human PKM. MS-identified peptides of PKM are highlighted in red. Black-labeled sequences belong to common regions of both PKM1 and PKM2 isoforms and green-labeled sequences identify isoform specificity. ( C ) Co-IP of recombinant (rec-) PSAT1 and PKM1 or PKM2. Immunocomplexes were precipitated using an anti-PSAT1 antibody and analyzed by immunoblot using anti-PKM1, anti-PKM2, and anti-PSAT1 antibodies. Recombinant proteins were used as input controls showing antibody specificity and PSAT1 alone was used as an IP control. Shown are representative images from two separate experiments.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Binding Assay, Silver Staining, Purification, Sequencing, Labeling, Co-Immunoprecipitation Assay, Recombinant, Western Blot

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Mutations within an isoform-specific region of PKM2 weakens the PSAT1 interaction. ( A ) Schematic representation of PKM2-specific mutations generated for the analysis of the PSAT1–PKM2 association. Ribbon representation of the structure of PKM2 colored by PKM1 sequence homology (right panel). Identical regions are shown in purple and divergent regions in cyan. The left panel depicts the site-directed mutagenesis of amino acids (MT1–4, highlighted in red) in the PKM2-specific region (denoted in the white box). ( B ) FLAG-PKM2 wild-type (WT), mutants (MT1–4) and FLAG-EV (-) were expressed in HEK293T cells. Endogenous PSAT1 protein complexes were immunoprecipitated and the association with PKM2 was assessed by immunoblot. A similar expression of FLAG-PKM2 variants is shown by immunoblot of FLAG fusion proteins from the protein lysate input with β-actin used for a protein loading control. Shown are representative images from three separate experiments. (-) denotes an empty vector.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Generated, Sequencing, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: PSAT1 associates with endogenous PKM2 in NSCLC cells but the loss of PSAT1 does not alter either PKM2 expression or pyruvate kinase activity. ( A ) Co-IP of PSAT1 and PKM2 in A549 and PC9 NSCLC cells. PSAT1-specific immunocomplexes were precipitated using anti-PSAT1 from a whole-cell lysate and analyzed for PKM2 by immunoblot with an anti-PKM2 antibody. Shown are representative images from three separate experiments. ( B ) Loss of PSAT1 expression in A549 and PC9 cells stably expressing PSAT1-specific shRNA. The PSAT1 expression was determined in whole-cell lysates from the control or shPSAT1-expressing cells by immunoblot using anti-PSAT1 and anti-α-tubulin (loading control). ( C ) Intracellular pyruvate kinase activity was determined in cell lysates from A549 or PC9 cells with or without PSAT1 expression. Data are represented as relative pyruvate kinase (PK) activity (control cells set to 1) and shown are the mean ± SD from four independent experiments. A statistical significance was determined by an unpaired t-test analysis. ( D ) Immunoblot analysis of PKM1 or PKM2 expression in whole-cell lysates from the control or PSAT1-silenced A549 and PC9 cells. Recombinant human PKM1 and PKM2 proteins were used as positive controls for antibody specificity and β-actin was used as a loading control. Shown are representative images from two separate experiments. IP: immunoprecipitation; IB: immunoblot; NS: not significant.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, Co-Immunoprecipitation Assay, Western Blot, Stable Transfection, shRNA, Recombinant, Immunoprecipitation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Silencing of PSAT1 suppresses the nuclear localization of PKM2 in EGFR activated NSCLC cells. ( A ) EGFR-mutant PC9 cells stably expressing the control or PSAT1 shRNA were treated with 1 µM of erlotinib. Cytoplasmic and nuclear fractions were examined by an immunoblot analysis using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three separate experiments. ( B ) Nuclear localization of PKM2 was examined in serum-starved PC9 cells expressing the control or PSAT1 shRNA by confocal microscopy. DAPI served as a control for nuclear staining. Arrowheads indicate nuclear PKM2 staining in representative images from three independent experiments. ( C ) Serum-starved A549 cells (EGFR wild-type) stably expressing control or PSAT1 shRNA were treated with or without EGF (100 ng/mL). Cytoplasmic and nuclear fractions were prepared and the PKM2 and PSAT1 localizations were analyzed by immunoblot. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three separate experiments.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Stable Transfection, Expressing, shRNA, Western Blot, Confocal Microscopy, Staining

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Re-expression of PSAT1 restores the nuclear localization of PKM2 and cell migration in silenced PC9 cells. ( A ) Immunoblot analysis for PKM2 and PSAT1 localization in PSAT1-silenced PC9 cells stably expressing an empty vector (EV) or FLAG-PSAT1. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( B ) Wound healing assay of the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells. Shown are representative images at 0 h and 24 h. The migrating cells are demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.005 and * p < 0.05. A.U.: arbitrary unit.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Migration, Western Blot, Stable Transfection, Plasmid Preparation, Wound Healing Assay

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Re-expression of nuclear-localized acetyl-mimetic (K433Q) PKM2, but not wild-type PKM2, partially rescues the migration defect due to the loss of PSAT1. ( A ) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing the nuclear-targeted wild-type PKM2 (FLAG-PKM2 NLS-WT ). Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2 NLS-WT -expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( B ) Wound healing assay of serum-starved PC9 cells expressing the control-EV, shPSAT1-EV, or shPSAT1-FLAG-PKM2 NLS-WT . Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. * p < 0.0001 and N.S.: not significant. ( C ) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing nuclear-targeted acetyl-mimetic (K433Q) PKM2. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2 NLS-K433Q -expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. ( D ) Wound healing assay of serum-starved PC9 cells expressing the control-EV or shPSAT1-EV and shPSAT1-FLAG-PKM2 NLS-K433Q . Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.0001 and * p < 0.05. EV: empty vector; A.U.: arbitrary unit; NLS: nuclear localization signal.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Migration, Western Blot, Stable Transfection, Wound Healing Assay, Plasmid Preparation

Journal: Cancers

Article Title: Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells

doi: 10.3390/cancers13163938

Figure Lengend Snippet: Schematic depicting putative nodes for PSAT1 regulation in the nuclear translocation and retention of PKM2 in response to EGFR activation. ( i ) PSAT1 interaction may promote cytoplasmic PKM2 phosphorylation and acetylation or suppress nuclear SIRT6-dependent deacetylation that contributes to nuclear PKM2 function in increasing pro-motility gene expression. ( ii ) PSAT1 may influence various steps involved in PKM2’s translocation and retention independent of direct PKM2 interaction. Dotted lines indicate putative PSAT1 regulatory steps.

Article Snippet: HEK293T cells were transfected with either FLAG-tagged empty vector (FLAG-EV) (FLAG-HA-pcDNA3.1 (Addgene, # 52535, Watertown, MA, USA)), PKM2 WT or mutant PKM2 (MT(1–4)) vectors using jetPEI (PolyPlus, New York, NY, USA) according to the manufacturer’s protocol.

Techniques: Translocation Assay, Activation Assay, Expressing