ds18561882  (MedChemExpress)

Journal: Nature Communications

Article Title: Mitochondrial one-carbon metabolism is required for TGF-β-induced glycine synthesis and fibrotic responses

doi: 10.1038/s41467-025-64320-2

Figure Lengend Snippet: a Western blot analysis of collagen and α-smooth muscle actin (α-SMA) protein expression in human lung fibroblasts (HLFs) transfected with siRNA targeting MTHFD2 or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. b Quantification of Collagen 1 and α-SMA levels in MTHFD2 knockdown HLFs relative to control cells at the indicated intervals. c Western blot analysis of GCN2 and S6-kinase phosphorylation and Collagen 1 expression in control and MTHFD2 knockdown HLFs. Cells were cultured in the presence or absence of extracellular glycine and were treated with TGF-β for the indicated intervals. Blots are representative of 4 separate experiments. d Western blot analysis of puromycin incorporation into newly translated proteins. Control and MTHFD2 knockdown HLFs were treated with TGF-β for 48 hours or left untreated. Cells were pulse labeled with puromycin for the indicated intervals. e qRT-PCR analysis of COL1A1 , ACTA2 , and COL3A1 mRNA expression in control and MTHFD2 knockdown HLFs either left untreated or treated with TGF-β for 48 hours. f Heatmap analysis of TGF-β-induced gene expression (differentially expressed genes between control knockdown untreated and control knockdown TGF-β, P < 0.01) in control and MTHFD2 knockdown HLFs treated with TGF-β for 48 hours. g Volcano plot showing differentially expressed genes between control knockdown TGF-β and combined MTHFD2 knockdown TGF-β-treated HLFs. h Proliferation analysis of control and MTHFD2 knockdown HLFs. i , j Analysis of cellular migration of control and MTHFD2 knockdown HLFs. k , l Analysis of contraction of control and MTHFD2 knockdown HLFs embedded in agarose and treated with TGF-β for 24 hours before gel release. All plots are presented as mean ± SD. Significance was calculated by 1-way ANOVA with Dunnett’s post-test ( b , n = 3 independent experiments), ( h , n = 4 biological replicates), ( j , n = 16 biological replicates), ( l , n = 4 biological replicates), 2-way ANOVA with Tukey’s post-test ( e , n = 3 biological replicates), and quasi-likelihood F-test ( f , n = 3 biological replicates). Source data are provided as a Source Data file.

Article Snippet: Mice receiving MTHFD2 inhibitor were IP injected with DS18561882 (MedChemExpress, HY-130251) beginning on day 8 after final bleomycin instillation.

Techniques: Western Blot, Expressing, Transfection, Knockdown, Control, Phospho-proteomics, Cell Culture, Labeling, Quantitative RT-PCR, Gene Expression, Migration

Journal: Nature Communications

Article Title: Mitochondrial one-carbon metabolism is required for TGF-β-induced glycine synthesis and fibrotic responses

doi: 10.1038/s41467-025-64320-2

Figure Lengend Snippet: a Schematic representation of metabolite labeling downstream of 2,3,3-D3-Serine. Human lung fibroblasts (HLFs) were transfected with siRNA targeting MTHFD2 or nontargeting siRNA. Cells were labeled with 2,3,3-D3-Serine and treated with TGF-β for 48 hours or left untreated. b – g Gas chromatography/mass spectrometry analysis of cellular metabolites. b Analysis of cellular glycine labeling from 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. c Relative levels of M + 1 glycine from ( b ). d Analysis of cellular serine after labeling with 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. e Relative levels of M + 1 and M + 2 serine from ( d ). f Analysis of cellular proline labeling from 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. g Relative levels of M + 1 and proline from ( f ). h Relative levels of media formate content in NHLFs treated with TGF-β or left untreated. i – k Liquid chromatography/mass spectrometry analysis of cellular metabolites. i Analysis of cellular GTP labeling from 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. j Analysis of cellular ATP labeling from 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. k Analysis of cellular SAM labeling from 2,3,3-D3-Serine in control and MTHFD2 knockdown HLFs. Bar graphs represent mean ± SD, n = 3 biological replicates ( b – g , i – k ), n = 4 biological replicates ( h ). Significance was calculated by 2-way ANOVA with Tukey’s post-test. Source data are provided as a Source Data file.

Article Snippet: Mice receiving MTHFD2 inhibitor were IP injected with DS18561882 (MedChemExpress, HY-130251) beginning on day 8 after final bleomycin instillation.

Techniques: Labeling, Transfection, Gas Chromatography, Mass Spectrometry, Control, Knockdown, Liquid Chromatography

Journal: Nature Communications

Article Title: Mitochondrial one-carbon metabolism is required for TGF-β-induced glycine synthesis and fibrotic responses

doi: 10.1038/s41467-025-64320-2

Figure Lengend Snippet: a Uniform manifold approximation and projection (UMAP) clustering of lineage labeled alveolar fibroblast populations from mice as annotated by Tsukui et al. . b UMAP representation of the expression of Mthfd2 in lineage labeled alveolar fibroblasts at the indicated day after bleomycin instillation. c Dot plot representation of the expression of 1 C metabolic enzymes in total lineage labeled alveolar fibroblasts at the indicated day after bleomycin instillation. d Dot plot representation of the expression of 1 C metabolic enzymes in fibroblast subpopulations as in ( a ) on Day 21 after bleomycin instillation. e UMAP clustering of lung fibroblast populations from 10 control lungs and 20 lungs from patients with pulmonary fibrosis as defined by Habermann et al. . f UMAP representation of the expression of MTHFD2 in human lung fibroblasts as in ( e ). g Dot plot representation of the expression of 1 C metabolic enzymes in fibroblast subpopulations as in ( e ). h UMAP representation of subclustered alveolar fibroblasts and myofibroblasts from ( e ) showing alveolar, inflammatory, and fibrotic groups. i UMAP representation of the expression of MTHFD2 in human lung fibroblasts as in ( h ). j Dot plot representation of the expression of 1 C metabolic enzymes in fibroblast subpopulations as in ( h ). k Histological analysis of Mthfd2 protein expression in mouse lung tissue 21 days after instillation of either bleomycin or saline (vehicle). l Histological analysis of MTHFD2 protein expression in lung tissue from a control donor, or a patient with IPF. m Pearson correlation of MTHFD2 gene expression with two independent measures of lung function (FVC-pre-BD and DLCO) in patients with IPF. Chart represents best fit linear regression with 95% confidence interval. Data is from GSE32537 .

Article Snippet: Mice receiving MTHFD2 inhibitor were IP injected with DS18561882 (MedChemExpress, HY-130251) beginning on day 8 after final bleomycin instillation.

Techniques: Labeling, Expressing, Control, Saline, Gene Expression