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: Folate shifts toward mitochondrial metabolism for HSC activation. A , when LX-2 cells were fed [2,3,3- 2 H] serine, thymidine triphosphate (dTTP) with 2 H 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- 2 H]-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- 2 H]]-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, Flow 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: 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, MTHFD1 LX-2 cells treated with TGF-β1(5 ng/ml) for 36 h. I , LX-2 cells were stimulated multiple times by TGF-β1(5 ng/ml) at 0 h, 24 h, 48 h and were pretreated with DMSO or SHIN1 (5 μM) or LY345899 (5 μM) at 24 h. Flow cytometry analysis for collagen α1(I) expression at 0 h, 24 h, 48 h, 72 h. J , knockdown SHMT2 and MTHFD2 LX-2 cells were stimulated multiple times by TGF-β1 (5 ng/ml) at 0 h, 24 h, 48 h. Flow cytometry analysis for collagen α1(I) expression at 0, 24, 48, 72 h. The data are shown as mean ± SEM. The statistical significance (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001) was tested by unpaired t test. 1C, one-carbon; FA, folic acid; HSC, hepatic stellate cell; MTHFD, methylenetetrahydrofolate dehydrogenase; 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: Gene Expression, Western Blot, Expressing, Knockdown, Flow Cytometry

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: 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: 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 hydroxyproline 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: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A, B SHMT2 purified by Flag-IP was collected after in-gel digestion and used in LC-MS/MS analysis to search for the binding proteins of SHMT2. (A) Flag-SHMT2 was transfected into 293T cells for 24 h, isolated by coimmunoprecipitation, separated by SDS-PAGE and stained using Coomassie. (B) Tabular display of the number of tryptic peptides from each of the indicated proteins that copurified with SHMT2. C HCT116 cells transfected with Flag-SHMT2 were immunoprecipitated with FLAG-M2 beads, followed by western blotting for p53 and SHMT2. D Immunoprecipitation using an anti-p53 antibody (Do-1) was followed by western blotting with anti-SHMT2 or anti-p53 antibodies (ab32389, abcam). E Cytoplasmic p53 bound to SHMT2. HCT116 cells transfected with Flag-WT, nuclear (NES-) or cytoplasmic p53 (NLS-) were immunoprecipitated with FLAG-M2 beads followed by western blotting for FLAG and SHMT2. F, G The colocalization of SHMT2 and cytoplasmic p53. (F) Representative micrographs of HCT116 cells transfected with plasmids expressing WT, nuclear (NES-) and cytoplasmic p53 (NLS-). (G) Representative micrographs of HCT116 cells stained for SHMT2 and p53. Scale bar, 10 μm.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transfection, Isolation, SDS Page, Staining, Immunoprecipitation, Western Blot, Expressing

Journal: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A Effect of SHMT2 on the maturation of LC3. HCT116 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (Sh-1, Sh-2 or Sh-3) lentivirus for 72 h and selected with puromycin (1 mg/ml) to establish stable cell lines. The protein levels of endogenous SHMT2, p62, LC3 and β-actin (as the internal standard) were examined by western blotting using anti-Flag, anti-SHMT2, anti-p62, anti-LC3 and anti-β-actin antibodies, respectively. B GFP–LC3 puncta induced by SHMT2 knockdown. Control and SHMT2-sh stable HCT116 cell lines were transfected with a GFP–LC3 plasmid and cultured in complete medium for 24 hours. Scale bar, 10 μm. C The percentage of cells showing accumulation of GFP–LC3 in puncta (GFP–LC3 vac ) is shown (mean ± s.d., n = 3; ** P < 0.01). D Ultrastructural evidence of autophagic vacuolization induced by SHMT2 depletion. E The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.d.; ** P < 0.01). F Effect of SHMT2 on the maturation of LC3 in sw480 cells. HCT116 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (Sh-1 or Sh-2) lentivirus for 72 h and selected with puromycin (1 mg/ml) to establish stable cell lines. The protein levels of endogenous SHMT2, p62, LC3 and β-actin (as the internal standard) were examined by western blotting using anti-Flag, anti-SHMT2, anti-p62, anti-LC3 and anti-β-actin antibodies, respectively. G The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Infection, Stable Transfection, Western Blot, Transfection, Plasmid Preparation, Cell Culture

Journal: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A-C Effect of SHMT2 and p53 on the maturation of LC3. The protein levels of SHMT2, p53, p62, LC3 and β-actin (as the internal standard) were assessed by western blotting using anti-Flag, anti-p53, anti-p62, anti-LC3 and anti-β-actin antibodies, respectively. (A) HCT116 cells were infected with Scramble-sh (Control) or p53 knockdown (Sh) lentivirus for 72 h and transfected with Flag-SHMT2 for 24 h. (B) Wild-type (WT) or p53–/– HCT116 cells were transfected with Flag-SHMT2 for 24 h. (C) Control or SHMT2-Sh stable cell lines were transfected with Flag-WT, nuclear (NES-) and cytoplasmic p53 (NLS-) plasmids for 24 h. Scale bar, 10 μm. D GFP–LC3 puncta formation induced by SHMT2-sh or p53 mutants. Control and SHMT2-sh stable HCT116 or SW480 cell lines were transfected with Flag-WT, nuclear (NES-) cytoplasmic p53 (NLS-) or GFP–LC3 plasmids and cultured in complete medium for 24 h. E The percentage of cells showing accumulation of GFP–LC3 in puncta (GFP–LC3 vac ) is shown (mean ± s.d., n = 3; ** P < 0.01). F SHMT2 disrupted the binding of cytoplasmic p53 to HDM2. HCT116 cells transfected with HDM2, SHMT2 and Flag-cytoplasmic p53 (NLS-) were immunoprecipitated with FLAG-M2 beads, followed by western blotting for p53, HDM2 and SHMT2. G SHMT2 maintained the stability of cytoplasmic p53. Western blot analysis of lysates of cell lines with stable SHMT2 overexpression and knockdown transfected with Flag-WT, nuclear (NES-) and cytoplasmic p53 (NLS-) plasmids and treated with the translation inhibitor cycloheximide (CHX, 50 μg/ml) for the indicated hours. H, I HCT116 cells transfected with Flag-cytoplasmic p53 (NLS-), GFP-SHMT2 or GFP-HDM2 were immunoprecipitated with FLAG-M2 beads, followed by western blotting for p53 and GFP. (H) 5-FU disrupted the binding of cytoplasmic p53 to SHMT2. (I) 5-FU promoted the binding of cytoplasmic p53 to HDM2. J Western blot analysis of lysates of HCT116 cell lines transfected with Flag-cytoplasmic p53 (NLS-) or SHMT2 plasmids and treated with CHX for the indicated hours with or without 5-FU.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Western Blot, Infection, Transfection, Stable Transfection, Cell Culture, Binding Assay, Immunoprecipitation, Over Expression

Journal: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A SHMT2 promotes apoptosis and inhibits autophagy in response to 5-FU. Western blot analysis of lysates of HCT116 cell lines transfected with SHMT2 or infected with SHMT2-Sh virus and treated with 5-FU (10 μM) for 24 h. The protein levels of SHMT2, p62, LC3, cleaved-Caspase 3, PARP and β-actin (as internal standards) were assessed with anti-SHMT2, anti-p62, anti-LC3, anti-cleaved Caspase 3, anti-PARP and anti-β-actin antibodies, respectively. B, C The indicated cells were treated with 5-FU (2 μM), 3-methyladenine (3-MA, 10 mM) or chloroquine diphosphate salt (CQ, 20 μM) for four days and stained with 0.1% crystal violet (B) or analyzed for cell viability by an MTT assay (C), ** P < 0.01. (c-e) The xenograft experiment with Control or SHMT2 knockdown cells treated with 5-FU or CQ is described in the Methods section. D Xenograft tumours were harvested and photographed. E,F The quantification of the (e) average volume and (f) weight of the xenograft tumours is shown. Five tumours from individual mice were included in each group; ** P < 0.01. G Sample images of immunohistochemical and hematoxylin-eosin staining (HE) of xenograft tumours. Scale bar, 50 μm.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Western Blot, Transfection, Infection, Virus, Staining, MTT Assay, Immunohistochemical staining

Journal: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A The expression of SHMT2 in three GEO data sets (GSE39582, GSE24551 and GSE21510). B Representative IHC images of anti-SHMT2 staining in peritumoural and CRC tissues. Scale bar, 50 μm. C A total of 378 paired CRC tissues assessed by IHC are shown. D Among the 378 stage II-III CRC tissues, 37.04% exhibited low expression of SHMT2 (score <9), while 62.96% expressed high expression of SHMT2 (score ≥9). E Survival of patients with different levels of SHMT2 expression. DFS and OS stratified by SHMT2 expression in patients with stage II-III disease and treated with 5-FU–based chemotherapy. F, G Comparison of SHMT2 and LC3-II expression in CRC tissues. (F) Sample images of immunohistochemical staining. Scale bar, 50 μm. (G) Spearman’s r-coefficient test for the evaluation of correlations between the SHMT2 and lc3-II immunohistochemical expression status in CRC tissues. Negative ρ values indicate a negative relationship. The significance level is indicated by the P value.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Expressing, Staining, Comparison, Immunohistochemical staining

Journal: bioRxiv

Article Title: Loss of SHMT2 mediates 5-FU chemoresistance by inducing autophagy in colorectal cancer

doi: 10.1101/680892

Figure Lengend Snippet: A Immunohistochemical images of SHMT2, LC3, and p62 staining in CRC tissues from four selected patients (two with low SHMT2 expression and two with high SHMT2 expression) using the indicated antibodies. B Schematic of the patient-derived xenograft (PDX) model. C-E The xenograft experiments with 5-FU or CQ treatment are described in the Methods section. (C) Xenograft tumours were harvested and photographed. The quantification of the (D) average volume and (E) weight of the xenograft tumours is shown. Four tumours from individual mice were included in each group, ** P < 0.01. F Sample immunohistochemical images of xenograft tumours. Scale bar, 50 μm.

Article Snippet: Antibodies against LC3 (ABC929, Sigma; ab48394, Abcam), SHMT2 (NBP1-80755, Novus Biologicals USA), p62 (p0017, Sigma), p53 (DO-1, Santa Cruz; ab32389, Abcam) and actin (A1978, Sigma) were used.

Techniques: Immunohistochemical staining, Staining, Expressing, Derivative Assay

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: the expression level of SHMT2 mRNA in human different types of kidney cancer a-g The expression of SHMT2 in different types of kidney cancer datasets from Oncomine database; h The expression of SHMT2 in renal clear cell carcinoma patients from GEPIA database. A total of 523 renal clear cell carcinoma tissues (red bar) and 100 normal renal tissues (gray bar) were collected in this database

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques: Expressing

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: Association between SHMT2 gene expression and clinical pathological parameters in patients with renal clear cell carcinoma (UALCAN). a expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ age; b expression of SHMT2 in normal and renal clear cell carcinoma tissues based on cancer stages; c expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ gender; d expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ race; e expression of SHMT2 in normal and renal clear cell carcinoma tissues based on tumor grade. #P:compared to normal group; &P: compared to the age (21–40 years) group; $P: compared to the Stage 2 group; *P: compared to the Caucasian group

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques: Gene Expression, Expressing

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: The prognostic value of SHMT2 mRNA in kidney cancer patients (K-M plotter) and the IHC of SHMT2 in kidney cancer (HPA). a, b Survival curve of SHMT2 for renal papillary cell carcinoma and renal clear cell carcinoma, respectively. c, d : IHC of SHMT2 in kidney cancer from healthy and pathological tissues

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques:

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: Co-expression genes of SHMT2 in kidney cancer by cBioPortal

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques:

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: Analysis of SHMT2’s co-expressed genes in kidney cancer a regression analysis of the expression between SHMT2 and NDUFA4L2 in kidney cancer by using cBioPortal tool. b Western blot analysis and representative image of SHMT2 expression in A498 and ACHN cells 24 h after transfection of SHMT2 expression vector or the negative control. c NDUFA4L2 expression at mRNA level in A498 and ACHN cells 24 h after transfection of SHMT2 expression vector or the negative control. d Western blot assay and representative results of NDUFA4L2 expression at protein level in A498 and ACHN cells 24 h after transfection of SHMT2 expression vector or the negative control. n = 3; #p < 0.05, vs negative control

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Negative Control

Journal: Biological Research

Article Title: Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

doi: 10.1186/s40659-020-00314-2

Figure Lengend Snippet: Association between NDUFA4L2 gene expression and clinical pathological parameters in patients with renal clear cell carcinoma (UALCAN). a expression of NDUFA4L2 in normal and renal clear cell carcinoma tissues based on cancer stages; b expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ age; c expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ gender; d expression of SHMT2 in normal and renal clear cell carcinoma tissues based on patients’ race; e expression of SHMT2 in normal and renal clear cell carcinoma tissues based on tumor grade. #P:compared to normal group; $P: compared to the Stage 2 group

Article Snippet: Human SHMT2 cDNA ORF clone (cat. RC204239) and the empty control pCMV6-AC (cat. PS100020) were obtained from OriGene (Rockville, USA).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Targeting one-carbon metabolic vulnerabilities of metastasis with therapeutic potential

doi: 10.64898/2026.02.07.704548

Figure Lengend Snippet: ( A, B, C, D ) Seahorse analysis confirmed that overexpression of PHGDH wildtype (WT) or dominant active form (DA) in A375p cells enhances both basal (A) and compensatory glycolysis (B), ATP production (C), and OCR (D). C, empty vector control. The data represent three independent experiments. Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. ( E, F, G, H ) Seahorse analysis confirmed that PHGDH deficiency (sgRNA) by CRISPR/CAS9 sgRNA in A375sm cells reduces both basal (E) and compensatory glycolysis (F), ATP production (G), and OCR (H). C, empty vector control. The data represent three independent experiments. Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. ( I, J ) Representation of lung bioluminescence and fluorescence images to track A375sm-Akaluc tumor cells transfected with vector control (C) or CRISPR/CAS9 sgRNA for PHGDH (sgRNA) at 30 min (I) and 1 week (J) after tail vein injection by In Vivo Imaging System (IVIS). ( K ) Gross pulmonary metastases from human melanoma A375sm-Akaluc cells transfected with PHGDH sgRNA (sgRNA) in NSG mice by tail vein injection. C, empty vector. N=10. Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ( L ) The data showed the bioluminescence signals of tumor metastasis in NSG mice, tracked by the In Vivo Imaging System (IVIS), for 57 days after transplantation. C, the A375sm-Akaluc cells transfected with the control plasmid; sgRNA, the A375sm-Akaluc cells transfected with the CRISPR/CAS9 sgRNA for the PHGDH plasmid. Ten mice per group (N=10). Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ( M ) Gross pulmonary metastases from human melanoma A375sm-Akaluc cells transfected with PHGDH sgRNA (sgRNA) in NSG mice by tail vein injection. C, empty vector. N= 10. Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ( N ) Analyses of TCGA data show that overexpression of De novo serine synthesis (SSP) metabolism-related genes (PHGDH, NQO1, PSAT1, and SHMT2) is associated with a shorter time of survival in all tumor samples in TCGA data [p = 6.098e-4 by Logrank (Mantel-Cox) test].

Article Snippet: ENOPH1, shRNA for ENOPH1, ENOPH1 TALEN, ENOPH1 CRISPR/CAS9 sgRNA, PHGDH wt, Akaluc plasmid, PHGDH dominant active form, PHGDH CRISPR/CAS9 sgRNA, SHMT2, SHMT2-CD, SHMT2 CRISPR/CAS9 sgRNA, MTHFD2, and PSPH expressing plasmids were provided by Addgene (Cambridge, MA).

Techniques: Over Expression, Plasmid Preparation, Control, Two Tailed Test, CRISPR, Fluorescence, Transfection, Injection, In Vivo Imaging, Transplantation Assay