Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: MicroRNA-340 Inhibits Esophageal Cancer Cell Growth and Invasion by Targeting Phosphoserine Aminotransferase 1.

doi: 10.1159/000430361

Figure Lengend Snippet: Fig. 4. PSAT1 is a direct target of miR-340. (A) The putative miR-340-binding sequence in the 3′-UTR of PSAT1 mRNA is shown. Muta tions were introduced into the PSAT1 3′-UTR sequence at the complementary site for the seed region of miR-340. (B) Luciferase reporter as says show the miR-340-me diated suppression of the ac tivity of the wild-type PSAT1 3′-UTR luciferase, but not the mutant 3′-UTR luciferase, in HEK-293 cells. (C) Ectopic ex pression of miR-340 in EC109 and EC7906 cells downregu lated the mRNA expression of PSAT1. (D) PSAT1, GSK- 3β, p-GSK-3β (Ser9), Snail, Vimentin and E-cadherin protein levels were deter mined by western blotting. *, p < 0.05; **, p < 0.01.

Article Snippet: The following antibodies were used: p-GSK-3β, GSK-3β, Snail, E-cadherin, Vimentin (Cell Signaling Technology); and PSAT1 (Proteintech Group Inc, Chicago, IL).

Techniques: Binding Assay, Sequencing, Luciferase, Mutagenesis, Expressing, Western Blot

Journal: Cancer Research

Article Title: Exogenous and Endogenous Sources of Serine Contribute to Colon Cancer Metabolism, Growth, and Resistance to 5-Fluorouracil

doi: 10.1158/0008-5472.can-20-1541

Figure Lengend Snippet: Figure 1. APC status impacts serine synthesis gene expression in murine intestinal tumors. A, The expression of Phgdh, Psat1, and Psph was compared in ileal polyps from ApcMin/þ mice versus normal ileal mucosa from WT mice by qRT-PCR. Gene expression in polypswas calculated relative to normal mucosa (n ¼ 5 per group). , P < 0.01 compared with normal tissue for each gene. B, Expression levels of Phgdh, Psat1, and Psph were determined by RNA-seq in colonic tissue from shRen mice given doxycycline for 15 weeks (shRen þ dox), shApc mice given doxycycline for 15 weeks (shApc þ dox), and shApc mice 4 days following doxycycline removal (shApc off dox; n ¼ 4 per group). , P < 0.05; , P < 0.001 as indicated. C, Expression levels of Phgdh, Psat1, and Psph were determined by RNA-seq in colonic tissue from shApc/KrasG12D mice given doxycycline for 15 weeks and are shown along with data found in B from the shRen þ dox and shApc þ dox groups (n ¼ 4 per group). , P < 0.05; , P < 0.01; , P < 0.001 compared with the shRen group for each gene as indicated.

Article Snippet: The membranes were blotted with antibodies towards PSAT1 (Novus Biologicals, NBP1–32920), PHGDH (Sigma-Aldrich, SAB2101795), PSPH (Thermo Fisher Scientific, PA5–19113), Phospho-Histone H2AX (Cell Signaling Technology, #2577L) and b-actin (Sigma Aldrich, A3854).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Cancer Research

Article Title: Exogenous and Endogenous Sources of Serine Contribute to Colon Cancer Metabolism, Growth, and Resistance to 5-Fluorouracil

doi: 10.1158/0008-5472.can-20-1541

Figure Lengend Snippet: Figure 2. PSAT1 expression is increased in human colorectal neoplasia. A, A previously published gene expression profile of 32 pairs of adenoma and normal adjacent colon tissue was mined to determine expression of serine biosynthesis enzymes (34). , P < 0.001 comparing polyp to normal for each gene. B, The data in A are shown as relative expression of the ratio of adenoma over normal for each gene in each pair of samples. Data are ranked from highest to lowest expression. Black line indicates a value of 1. C, PSAT1 expression was assessed by IHC in tissue sections of colon adenomas (Ad) with normal adjacent tissue (N; 40; inset, 200). D, Paired colorectal normal and adenocarcinoma samples were probed for PSAT1 expression by Western blot analysis. b-Actin was used as a loading control. E, PSAT1 expression was assessed by IHC in tissue sections of colon adenocarcinomas (T) with normal adjacent tissue (N; 40; inset, 200).

Article Snippet: The membranes were blotted with antibodies towards PSAT1 (Novus Biologicals, NBP1–32920), PHGDH (Sigma-Aldrich, SAB2101795), PSPH (Thermo Fisher Scientific, PA5–19113), Phospho-Histone H2AX (Cell Signaling Technology, #2577L) and b-actin (Sigma Aldrich, A3854).

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: Cancer Research

Article Title: Exogenous and Endogenous Sources of Serine Contribute to Colon Cancer Metabolism, Growth, and Resistance to 5-Fluorouracil

doi: 10.1158/0008-5472.can-20-1541

Figure Lengend Snippet: Figure 3. Restriction of exogenous and endogenous sources of serine maximally suppresses colon cancer cell proliferation and alters metabolism. A, Western blotting of PHGDH, PSAT1, and PSPH following CRISPR-based deletion of PSAT1 in HCT116 and DLD1 human colon cancer cells containing Cas9, using three different guide RNAs. EV, empty vector. (Continued on the following page.)

Article Snippet: The membranes were blotted with antibodies towards PSAT1 (Novus Biologicals, NBP1–32920), PHGDH (Sigma-Aldrich, SAB2101795), PSPH (Thermo Fisher Scientific, PA5–19113), Phospho-Histone H2AX (Cell Signaling Technology, #2577L) and b-actin (Sigma Aldrich, A3854).

Techniques: Western Blot, CRISPR, Plasmid Preparation

Journal: Cancer Research

Article Title: Exogenous and Endogenous Sources of Serine Contribute to Colon Cancer Metabolism, Growth, and Resistance to 5-Fluorouracil

doi: 10.1158/0008-5472.can-20-1541

Figure Lengend Snippet: Figure 5. Combinationof PSAT1 deletion and removal of serine from diet suppresses the growth of colon tumor xenografts. EV and PSAT1KO HCT116 (A)and DLD1 (B) cellswere injected into the flanks of nude mice given complete amino acid–based diet or Ser Def diet and tumor volume was measured over time. , P < 0.05 compared with the other three conditions (A) or as indicated (B). n ¼ 9–10 tumors per condition. Tumor volume data at the terminal time point from A and B were evaluated for significant differences among the groups in HCT116 (C) and DLD1 (D) xenografts. , P < 0.01 compared with the other three conditions (C); , P < 0.05 as indicated (D).

Article Snippet: The membranes were blotted with antibodies towards PSAT1 (Novus Biologicals, NBP1–32920), PHGDH (Sigma-Aldrich, SAB2101795), PSPH (Thermo Fisher Scientific, PA5–19113), Phospho-Histone H2AX (Cell Signaling Technology, #2577L) and b-actin (Sigma Aldrich, A3854).

Techniques: Injection

Journal: Redox Biology

Article Title: Ferroptosis is governed by differential regulation of transcription in liver cancer

doi: 10.1016/j.redox.2019.101211

Figure Lengend Snippet: HBA1 and STMN1 regulate ferroptosis by affecting GSH by PSAT1. (A) Schematic presentation of signal pathways that affect accumulation of lipid ROS to cause ferroptosis. (B) Concentrations of MDA and GSH in control cells, HepG2 and Bel-7402 cells with HBA1 and STMN1 overexpressed or knocked down. (C) The metabolic axis from glucose to glycine and cysteine. (D) Concentration of p-Pyr and p-Ser in control cells, HepG2 and Bel-7402 cells with HBA1 and STMN1 overexpressed or knocked down. (E) Concentrations of MDA, GSH and p-Ser in control cells, HepG2 and Bel-7402 cells with PSAT1 knocked down in the presence or absence of simultaneous PSAT1 overexpression, with or without HBA1 or STMN1 overexpression, as indicated. The data are shown as the means + SD from three independent experiments. **, p < 0.01 indicate statistical significance. The data were analyzed by a one-way ANOVA test.

Article Snippet: The HBA1, STMN1, HIC1, HNF4A and PSAT1 expression plasmids were purchased from Origene (Beijing, China).

Techniques: Control, Concentration Assay, Over Expression

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: Loss of PSAT1 decreases cell migration in EGFR-mutant PC9 and EGF-stimulated A549 NSCLC cells. ( A ) Wound healing assay of PC9 cells expressing the control or PSAT1-specific shRNA. Shown are representative images taken at 0 h and 24 h. Migrating cells are demarcated by continuous white lines. Data are presented as a migrated area after 24 h and shown is the mean ± SE from three independent experiments. A statistical significance was determined by a paired t-test analysis. * is p < 0.005. ( B ) Boyden chamber migration assay on serum-starved A549 cells expressing the control or PSAT1 shRNA. A total of 100 ng/mL EGF serum-free media was used as a chemo-attractant and migrated cells were fixed and stained with crystal violet after 24 h. Shown are representative images of the migrated cells and quantification is demonstrated as the mean ± SE of the% migration area from three independent experiments. A statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparison test. * p = 0.0001. N.S.: not significant; A.U.: arbitrary unit.

Article Snippet: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Mutagenesis, Wound Healing Assay, Expressing, shRNA, 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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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: The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Activation Assay, Expressing

Journal: Cell Metabolism

Article Title: Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation

doi: 10.1016/j.cmet.2019.06.003

Figure Lengend Snippet:

Article Snippet: Mouse anti-PSAT1 , Novus Biologicals , Cat#H00029968-A01 RRID: AB_547380.

Techniques: Virus, Recombinant, Polymer, Protease Inhibitor, Membrane, Adhesive, Cell Viability Assay, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Modification, Software, Quantitative Proteomics, Sterility