shmt2 33443s Search Results


shmt2 33443s  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc shmt2 33443s
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 <t>SHMT2</t> 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.
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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.

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

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.

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

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.

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

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.

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