Journal: International Journal of Molecular Sciences

Article Title: Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

doi: 10.3390/ijms21051661

Figure Lengend Snippet: The effect of ethanol and curcumin on transcription of PKM1, PKM2, PHGDH and SHMT2 in four different cancer cell lines. One-way ANOVA with Tukey post-hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The sets of primers and probes for targets were: Hs00987255_m1 (PKM1); Hs00987262_g1 (PKM2); Hs01059260_g1 (SHMT2) and Hs00198333_m1 (PHGDH).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

doi: 10.3390/ijms21051661

Figure Lengend Snippet: Primers used in end-point PCR and quantitative PCR based on SYBR ® Green chemistry.

Article Snippet: The sets of primers and probes for targets were: Hs00987255_m1 (PKM1); Hs00987262_g1 (PKM2); Hs01059260_g1 (SHMT2) and Hs00198333_m1 (PHGDH).

Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Amplification

Journal: Cell reports

Article Title: Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion

doi: 10.1016/j.celrep.2020.108375

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RNA was reverse transcribed using the Bio-Rad iScript cDNA synthesis kit. qPCR was performed with Applied Biosystems TaqMan® gene expression assays for SHMT1 (Rn01751636_m1), SHMT2(Rn01768052_g1), SDH (Rn01499872_m1), CPS1 (Rn00567109_m1), OTC (Rn00565169_m1), ASS1 (Rn00565808_g1), ASL (Rn01480437_g1), ARG1 (Rn00691090_m1), and PPIA (Rn00690933_m1) on a Viia 7 Real-Time PCR system (Applied Biosystems).

Techniques: Recombinant, Software

Journal: Nature Communications

Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

doi: 10.1038/s41467-018-04602-0

Figure Lengend Snippet: Key driver MTHFD2 and oxPAPC induce endothelial Bayesian amino acid subnetwork. a Network view of the oxPAPC Bayesian subnetwork with MTHFD2 as key driver. Nodes that belong to indicated significantly overrepresented canonical gene set categories are highlighted respectively. Node size reflects out-degree. b Schematic diagram of enzymes within the MTHFD2 network (blue) that are directly or indirectly involved in serine, glycine, cysteine, methionine, aspartate, and asparagine (orange) interconversion as well as interconversion of tetrahydrofolates (THF) inside mitochondria (green). CBS: Cystathionine-Beta-Synthase, PCK2: Phosphoenolpyruvate Carboxykinase 2, GOT1 Glutamic-Oxaloacetic Transaminase 1, GLDC: Glycine Decarboxylase, ASNS: Asparagine Synthetase. c−l Experimental validation of the MTHFD2 network as assessed by quantitative RT-PCR. HAEC with and without knockdown of the key driver MTHFD2 or the downstream node PSAT1 were exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC for 4 h ( n ≥ 4). Genes belonging to the MTHFD2 network are framed by the color of the corresponding gene set category as in a . Data are represented as mean ± SEM, * p ≤ 0.05 ( MTHFD2 or PSAT1 vs Control siRNA), # p ≤ 0.05 (oxP vs Ct) (ANOVA with Bonferroni post-hoc test). MTHFD2: Methylenetetrahydrofolate Dehydrogenase (NADP + Dependent) 2-Methenyltetrahydrofolate Cyclohydrolase, SHMT2: serine hydroxymethyltransferase 2, PHGDH: phosphoglycerate dehydrogenase, PSAT1: phosphoserine aminotransferase 1, CEBPB: CCAAT/Enhancer Binding Protein Beta, GARS: Glycyl-TRNA Synthetase, CARS Cysteinyl-TRNA Synthetase, SLC7A5: Solute Carrier Family 7 Member 5, SLC7A1: Solute Carrier Family Member 1, MTHFD1L: Methylenetetrahydrofolate Dehydrogenase (NADP + Dependent) 1-Like

Article Snippet: The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Knockdown, Control, Binding Assay

Journal: Nature Communications

Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

doi: 10.1038/s41467-018-04602-0

Figure Lengend Snippet: MTHFD2 is deregulated in cardiovascular disease. a Glycine to serine ratio in plasma of human subjects with no atherosclerotic plaque (NP) ( n = 26), stable atherosclerotic plaque (SP) ( n = 26), and unstable atherosclerotic plaque (UP) ( n = 26) as assessed by mass spectrometry. b Scatter plots showing expression correlation in 126 human carotid plaque samples between MTHFD2 and genes of the MTHFD2 network (colored according to Fig. ) as well as Nrf2 ( NFE2L2 ) and ATF4 as calculated by Pearson correlation. c , d Relative mRNA expression of MTHFD2 and SHMT2 in plaque material of human subjects with unstable atherosclerotic plaque (UP) ( n = 20), stable atherosclerotic plaque (SP) ( n = 20), or non-atherosclerotic artery (NP) ( n = 8) (normalized to 18SrRNA ) ( n = 8). e , f Western blot analysis of MTHFD2 expression ( e ) and quantification ( f ) of plaque material from human subjects with unstable atherosclerotic plaque (SP) ( n = 20), stable atherosclerotic plaque (SP) ( n = 20), or non-atherosclerotic artery (NP) ( n = 8). g – j Relative mRNA expression of Mthfd2 , Phgdh , Shmt2 , and Slc3a2 in mouse thoracic aortic rings kept in organ culture and exposed to medium (1% FCS) with or without oxPAPC and rapamycin as indicated for 8 h (normalized to 18SrRNA ) ( n ≥ 4). k , l Relative mRNA expression of Mthfd2 and Shmt2 in the endothelium of partially ligated left carotid artery (LCA) compared to healthy right carotid artery (RCA) 48 h post ligation (normalized to 18S rRNA ) ( n = 3). (* p ≤ 0.05 Student’s t test). m Relative mRNA expression of Mthfd2 in the endothelium of the left carotid artery of ApoE−/− mice which were fed with high fat diet (HFD) for 0, 1, or 4 days (normalized to 18S rRNA ) ( n = 5). n – q Relative mRNA expression of MTHFD2 , PHGDH , CEBPB and PCK2 in HAEC exposed to HDL from healthy human subjects ( n = 10) or human subjects with CAD ( n = 10) for 4 h. (* p ≤ 0.05 Mann Whitney test). r , s Western blot analysis of MTHFD2 ( r ) and quantification ( s ) of HAEC treated as in f for 24 h ( n = 10) (* p ≤ 0.05 Mann Whitney test). Data are represented as mean ± SEM, * p ≤ 0.05 (ANOVA with Newman−Keuls post-hoc test if not otherwise indicated)

Article Snippet: The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500).

Techniques: Clinical Proteomics, Mass Spectrometry, Expressing, Western Blot, Organ Culture, Ligation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

doi: 10.1038/s41467-018-04602-0

Figure Lengend Snippet: Association between genes within the MTHFD2 network and CAD risk loci

Article Snippet: The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500).

Techniques:

Journal: The Journal of biological chemistry

Article Title: Mitochondrial folate metabolism-mediated α-linolenic acid exhaustion masks liver fibrosis resolution.

doi: 10.1016/j.jbc.2023.104909

Figure Lengend Snippet: Figure 3. Folate shifts toward mitochondrial metabolism for HSC activation. A, when LX-2 cells were fed [2,3,3-2H] serine, thymidine triphosphate (dTTP) with 2H was determined by mass spectrometry. dTTP with one deuterium (M + 1) is produced via the mitochondrial folate metabolism through SHMT2 and with two deuteriums (M + 2) via the cytosolic SHMT1. B, the percentage labeling of intracellular glycine when LX-2 were fed [2,3,3-2H]-serine in high (2 μM) and physiological (200 nM) FA levels. C, the ratio of dTTP M+1 and M+2 when LX-2 cells were fed [2,3,3-2H]]-serine in high and physiological FA levels. D, the subcellular distribution of Cy5-labeled folate in primary murine HSCs with TGF-β1(5 ng/ml) treatment for 36 h (scale bars represent 10 μm). E, flow cytometry analysis of FA-FITC in LX-2 cells after stimulation with control or TGF-β1(5 ng/ml) for 36 h, then treated with FA-Cy5 for 30 min. F, mRNA levels of SLC25A32 in LX-2 cells after treatment with TGF-β1(5 ng/ml) for 24 h. G, knockdown efficiency of three independent SLC25A32 siRNA in LX2 cells. H, immunoblotting for α-SMA and collagen α1(I) proteins expression in knockdown SLC25A32 LX-2 cells treatment with TGF-β1 (5 ng/ml) for 36 h. I, Western blot performed on cell lysates for phosphorylated SMAD2/3 in knockdown SLC25A32 LX-2 cells treated with or without TGF-β1 (5 ng/ml) for 30 min. The statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was tested by student’s unpaired t test. FA, folic acid; HSC, hepatic stellate cell; SHMT, serine hydroxymethyltransferase.

Article Snippet: Antibodies were purchased from the following resources: antibodies to phospho-SMAD2/3 (8828S), SMAD2/3 (8685S), SHMT1 (80715S), SHMT2 (33443S) from Cell Signaling Technology; antibodies to α-Actin (1A4) (ab150301) and TGFBR1(ab31013) from Abcam; antibodies to MTHFD1 (67670-1-1g), MTHFD1L (16113-1-AP) from Proteintech; antibodies to collagen 1α(I) from Southern Biotech; antibodies to GAPDH (AM1021B), Tubulin (M20005M) from Abmart; antibodies to α-Actin (1A4) (SC-32251) from Santa Cruz Biotechnology; antibody to MTHFD2 (GTX104990-S) from Gene Tex.

Techniques: Activation Assay, Mass Spectrometry, Produced, Labeling, Cytometry, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of biological chemistry

Article Title: Mitochondrial folate metabolism-mediated α-linolenic acid exhaustion masks liver fibrosis resolution.

doi: 10.1016/j.jbc.2023.104909

Figure Lengend Snippet: Figure 4. SHMT2-/MTHFD2-mediated mitochondrial folate metabolic pathway sustains TGF-β1 signaling. A, compartmentalization of FA-mediated 1C metabolism. B, qPCR detection of one-carbon metabolism gene expression in primary human HSC datasets from GSE67664. C, Western blot performed on cell lysates for α-SMA and collagen α1(I) after LX-2 cells were pretreated with DMSO or SHIN1 (1.25, 2.5, 5, 10 μM) or LY345899 (2.5, 5, 10, 20 μM) for 6 h, then stimulated with TGF-β1(5 ng/ml) for 24 h. D-H, Western blot for collagen α1(I) proteins expression in knockdown SHMT2, MTHFD2, MTHFD1L, SHMT1,

Article Snippet: Antibodies were purchased from the following resources: antibodies to phospho-SMAD2/3 (8828S), SMAD2/3 (8685S), SHMT1 (80715S), SHMT2 (33443S) from Cell Signaling Technology; antibodies to α-Actin (1A4) (ab150301) and TGFBR1(ab31013) from Abcam; antibodies to MTHFD1 (67670-1-1g), MTHFD1L (16113-1-AP) from Proteintech; antibodies to collagen 1α(I) from Southern Biotech; antibodies to GAPDH (AM1021B), Tubulin (M20005M) from Abmart; antibodies to α-Actin (1A4) (SC-32251) from Santa Cruz Biotechnology; antibody to MTHFD2 (GTX104990-S) from Gene Tex.

Techniques: Gene Expression, Western Blot, Expressing, Knockdown

Journal: The Journal of biological chemistry

Article Title: Mitochondrial folate metabolism-mediated α-linolenic acid exhaustion masks liver fibrosis resolution.

doi: 10.1016/j.jbc.2023.104909

Figure Lengend Snippet: Figure 5. Mitochondrial folate metabolism induces ALA exhaustion in activated HSCs. A, D, and G, differential metabolites abundance in LX-2 cells transfected with siNC, siSHMT2, or siSLC25A32 24 h after medium or TGF-β1 (5 ng/ml) stimulation (n = 3). B, E, and H, metabolite set enrichment analysis in LX2 cells transfected with siNC, siSHMT2, or siSLC25A32 24 h after medium or TGF-β1 (5 ng/ml) stimulation (n = 3). C, F, and I, heatmap of metabolites in LX-2 cells transfected with siNC, siSHMT2, or siSLC25A32 24 h after medium or TGF-β1 (5 ng/ml) stimulation (n = 3). J, LC-MS analysis of intracellular alpha-linolenic acid in knockdown SHMT2 or SLC25A32 LX-2 cells after treatment with TGF-β1 (5 ng/ml) for 24 h (n = 4). The statistical significance (*p < 0.05, **p < 0.01) was tested by student’s unpaired t test. ALA, α-linolenic acid; HSC, hepatic stellate cell; SHMT, serine hydroxymethyltransferase.

Article Snippet: Antibodies were purchased from the following resources: antibodies to phospho-SMAD2/3 (8828S), SMAD2/3 (8685S), SHMT1 (80715S), SHMT2 (33443S) from Cell Signaling Technology; antibodies to α-Actin (1A4) (ab150301) and TGFBR1(ab31013) from Abcam; antibodies to MTHFD1 (67670-1-1g), MTHFD1L (16113-1-AP) from Proteintech; antibodies to collagen 1α(I) from Southern Biotech; antibodies to GAPDH (AM1021B), Tubulin (M20005M) from Abmart; antibodies to α-Actin (1A4) (SC-32251) from Santa Cruz Biotechnology; antibody to MTHFD2 (GTX104990-S) from Gene Tex.

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Knockdown

Journal: The Journal of biological chemistry

Article Title: Mitochondrial folate metabolism-mediated α-linolenic acid exhaustion masks liver fibrosis resolution.

doi: 10.1016/j.jbc.2023.104909

Figure Lengend Snippet: Figure 7. Knockdown of SHMT2/MTHFD2 promotes liver fibrosis resolution in NASH mice. A, schematic illustrating animal experimental design. B and C, liver Shmt2 and Mthfd2 mRNA levels. Ad-shRNA–medicated Shmt2 and Mthfd2 knockdown in mouse liver tissue (n = 4). D, schematic representation of vitamin A–coupled liposomes nanoparticle for the delivery of siRNA. E, representative image of H&E, Masson, and sirius red–stained paraffin-embedded sections of mouse liver tissues (scale bar represents 50 μm). F, Ishak fibrosis stage scores at indicated treatment conditions (n = 6). G, hepatic hydroxy- proline content (n = 6). H and I, ALT and AST levels of plasma (n = 6). J and K, hepatic mRNA expression of fibrogenic genes Acta2 and Col1a1 (n = 5). The statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was tested by student’s unpaired t test. AST, aspartate aminotransferase; ALT, alanine aminotransferase; H&E, hematoxylin-eosin staining; MTHFD, methylenetetrahydrofolate dehydrogenase; NASH, nonalcoholic steatohepatitis; SHMT, serine hydroxymethyltransferase.

Article Snippet: Antibodies were purchased from the following resources: antibodies to phospho-SMAD2/3 (8828S), SMAD2/3 (8685S), SHMT1 (80715S), SHMT2 (33443S) from Cell Signaling Technology; antibodies to α-Actin (1A4) (ab150301) and TGFBR1(ab31013) from Abcam; antibodies to MTHFD1 (67670-1-1g), MTHFD1L (16113-1-AP) from Proteintech; antibodies to collagen 1α(I) from Southern Biotech; antibodies to GAPDH (AM1021B), Tubulin (M20005M) from Abmart; antibodies to α-Actin (1A4) (SC-32251) from Santa Cruz Biotechnology; antibody to MTHFD2 (GTX104990-S) from Gene Tex.

Techniques: Knockdown, shRNA, Liposomes, Staining, Clinical Proteomics, Expressing

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: SHMT2 was overexpressed and associated with tumor progression in HNC. ( A ) SHMT2 was expressed significantly more highly in tumor tissues than in normal tissues of HNC in The Cancer Genome Atlas (TCGA) database ( p < 0.0001). ( B ) Patients from TCGA with low SHMT2 expression had a better survival rate than those with high SHMT2 expression ( p = 0.012). ( C – H ) Expression of SHMT2 in HNC based on the TCGA database according to sex, histological grade ( G ), lymph node metastasis ( N ), distant metastasis ( M ), TNM (primary tumor, lymph node, distant metastasis) stage, and lymphovascular invasion. ( I – K ). The SHMT2 levels of three HNC tissue samples and their adjacent normal tissue samples were examined by immunohistochemistry. p < 0.05 was recognized as statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: SHMT2 played an oncogenic role and regulated the epithelial–mesenchymal transition (EMT) in HNC cells. ( A , B ) Normal head and neck cell lines (hFB) and HNC cell lines (FADU, SNU1041, SNU1076, SCC15, SCC25, HEP-2, and YD8) were subjected to reverse-transcription PCR analysis and Western blot analysis. ( C , D ) FADU and SNU1041 cells were infected with scramble-sh (shNC) or SHMT2 knockdown (shSHMT2-1, shSHMT2-2) lentivirus for 2 weeks. The stable cell lines were selected with puromycin (1 mg/mL), and cell viability was analyzed using the WST-1 assay. The levels of SHMT2 were detected by Western blots. ( E , F ) Representative images of colony formation assays from FADU and SNU1041 stable cell lines and quantitative analysis of colony formation assays. ( G , H ) FADU and SNU1041 cells transfected with shSHMT2-1 and shSHMT2-2 displayed significantly lower migration and invasion capacity than those transfected with each negative control (shNC). ( I , J ) Differences in the expression of EMT markers among FADU and SNU1041 cells transfected with shSHMT2 or shNC were detected by Western blotting. Data were presented as mean ± SD of three independent experiments. Differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Western Blot, Infection, Stable Transfection, WST-1 Assay, Transfection, Migration, Negative Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: GO and KEGG enrichment analyses of DEGs between the low and high SHMT2 in the TCGA HNC cohort. Genes with |log2Fold Change|> 1 and a p -value < 0.01 were defined as differentially expressed genes (DEGs). The up-regulated genes are marked in red, and the down-regulated genes are marked in blue. ( A ) Volcano plot of DEGs. ( B , C ) GO enrichment and KEGG pathway analysis results for DEGs. ( D ) The GSEA heat map shows differences in the expression of the core enrichment genes involved in the stem cell maintenance gene set between the patients with high and low SHMT2 expression. ( E ) Correlation between POU5F1 ( OCT4 ) and SHMT2 , ( F ) Correlation between SOX2 and SHMT2 , ( G ) Correlation between ALDH1A1 and SHMT2 , and ( H ) Correlation between FGF19 and SHMT2 . Differences were considered relevant at p < 0.05.

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: Silencing SHMT2 attenuated stemness properties in FADU and SNU1041 cells. ( A , B ) Compared with the control group, SHMT2 mRNA expression was significantly decreased in the sh SHMT2 -transfected FADU and SNU1041 cells; the mRNA expression of OCT4, SOX2 , and NANOG was also significantly decreased after silencing SHMT2. ( C , D ) The protein expression of OCT4, SOX2, and NANOG was also significantly decreased in sh SHMT2 -transfected FADU and SNU1041 cells. ( E ) Representative images of tumor spheres in shNC- and shSHMT2-transfected FADU cells (scale bar: 100 μm). ( F ) A comparison of the number of spheres between shNC and sh SHMT2 -transfected FADU cells. Spheres were counted after 10 days of incubation. The sphere number of each group was normalized to the shNC group. ( G ) Comparison of sphere diameters. ( H ) Representative images of tumor spheres in shNC- and shSHMT2-transfected SNU1041 cells (scale bar: 100 μm). ( I ) Comparison of the number of spheres between the shNC and sh SHMT2 -transfected SNU1041 cells. The sphere number of each group was normalized to the shNC group. ( J ) Comparison of sphere diameters in SNU1041 cells. ( K ) The protein expression of OCT4, SOX2, and NANOG was significantly decreased in sh SHMT2 -transfected FADU sphere-forming cells. Differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001). All experiments were repeated at least three times.

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Expressing, Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: SHMT2 promoted stem cell-like properties by activating the Notch and Wnt signaling pathways in HNC. ( A – F ) Notch- and Wnt pathway-related gene sets were enriched in HNC patients with high SHMT2 expression in the TCGA dataset. ( G – J ) Reverse-transcription quantitative PCR analysis of Notch and Wnt pathway-related genes with SHMT2 knockdown in FADU and SNU1041 cells. ( K , L ) Western blot analysis of Notch and Wnt pathway-related proteins with SHMT2 knockdown in the FADU and SNU1041 cells. Differences were considered significant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SHMT2 Induces Stemness and Progression of Head and Neck Cancer

doi: 10.3390/ijms23179714

Figure Lengend Snippet: Tumor growth in a mouse xenograft model after SHMT2 knockdown. 2 × 10 6 control-transfected Luciferase-expressing FADU (FADU-Luc) cells (shNC) and shRNA-transfected FADU-Luc cells (shSHMT2-1 and sh SHMT2 -2) were subcutaneously injected into 15 BALB/c-nude mice. ( A , B ) Representative final tumor images of cancer cells tracked with the in vivo imaging system following the injection of mice with FADU-Luc cells. ( C – E ) Images of tumors at the experimental endpoint. The tumors weight and volume were measured at the time of sacrifice. ( F ) Western blots from xenograft tumor tissues. Changes in the protein expression of SHMT2 , OCT4 , SOX2 , NANOG , E-cadherin, and N-cadherin in the xenograft tissues of each group. ( G ) Representative images of immunohistochemical staining of the negative control group and sh SHMT2 cell-injected groups. Scale bar, 100 μm. Results were analyzed using one-way analysis of variance. Differences were considered significant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: For the experiments of SHMT2 overexpression, a pCMV6-empty-myc-DDK tagged plasmid (control, PS100001) and a pCMV6 SHMT2-myc-DDK tagged plasmid ( SHMT2, RC204239) were purchased from OriGene.

Techniques: Transfection, Luciferase, Expressing, shRNA, Injection, In Vivo Imaging, Western Blot, Immunohistochemical staining, Staining, Negative Control

Journal: Molecular cancer therapeutics

Article Title: Repurposing the antidepressant sertraline as SHMT inhibitor to suppress serine/glycine synthesis addicted breast tumor growth

doi: 10.1158/1535-7163.MCT-20-0480

Figure Lengend Snippet: (A) PHGDH enzymatic in vitro assay, measuring PHGDH activity upon addition of indicated concentrations of sertraline (left) and thimerosal (right). Values are presented relative to the control (n = 3). (B) SHMT1 (left) and SHMT2 (right) in complex with sertraline (grey), with a magnified view of the binding pocket showing the interactions formed by sertraline. H-bonds formed by sertraline are presented as yellow dashes. The known SHMT inhibitor SHIN1, with a pyrazolopyran scaffold, is shown in magenta. (C) Schematic overview of isotopic tracing with [2,3,3-2H]-serine, showing 2H incorporation in downstream metabolites glycine and thymidine (dTTP). Cells taking up fully deuterated (M+3) serine use this to synthesize glycine with one deuterium label (M+1). While cytosolic methylene-THF production, by SHMT1, results in double 2H-labeled (M+2) dTTP (red dots), mitochondrial SHMT2 will generate single 2H-labeled (M+1) dTTP (blue dots). (D) Serine mass distribution showing the labeling fraction of each mass upon treatment of MDA-MB-468 cells with control (DMSO) or sertraline (5 μM) for 48 hours (n = 3, Multiple t-test). (E) Deuterium M+1 labeled dTTP fraction in MDA-MB-468 cells treated with control (DMSO) or sertraline (5 μM) for 48 hours. Values are presented relative to the control (n = 3, Student’s t-test). (F) Fluorescence distribution curves (left) and dose response curve (right) for SHMT2 incubated with different concentrations of sertraline (0.36 μM - 730 μM). Kd = 13.1 μM. Error bars represent the standard deviation of the measurements of two independent repeats. (G) Melting temperature (Tm) curves demonstrating sertraline-induced destabilization of SHMT1. One representative result of two biological replicates is shown. (H) Serine and glycine uptake (-) and secretion (+) rates (AU/cell/h) of MDA-MB-468 cells treated with indicated concentrations of sertraline for 72 h (n = 3, One-way ANOVA, Dunnett’s multiple comparisons). In (A, D-F and H) data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant human SHMT2 (OriGene #TP760127) was fluorescently labeled using the Monolith NT protein labeling kit Red-NHS (NanoTemper Technologies).

Techniques: In Vitro, Activity Assay, Binding Assay, Labeling, Fluorescence, Incubation, Standard Deviation

Journal: Molecular cancer therapeutics

Article Title: Repurposing the antidepressant sertraline as SHMT inhibitor to suppress serine/glycine synthesis addicted breast tumor growth

doi: 10.1158/1535-7163.MCT-20-0480

Figure Lengend Snippet: Computational docking of sertraline to SHMT1 and SHMT2. Docking scores for sertraline into the active pocket of SHMT1 and SHMT2. The already identified dual SHMT1/2 inhibitor SHIN1, based on a pyrazolopyran scaffold, was used as reference structure.

Article Snippet: Recombinant human SHMT2 (OriGene #TP760127) was fluorescently labeled using the Monolith NT protein labeling kit Red-NHS (NanoTemper Technologies).

Techniques: