Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: Primer sequences used for real-time PCR.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Sequencing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A expression was upregulated in inflamed gingival tissues.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A inhibition decreased P. gingivalis -induced inflammatory responses in hGfs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Inhibition

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A-knockdown reduced inflammation in P. gingivalis -infected hGFs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Knockdown, Infection

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: NF-κB/MAPK pathway was involved in the enhanced MAT2A expression.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: PERK signaling regulates MAT2A via ATF4 to support enhanced methionine metabolism. (A) KEGG enrichment analysis of upregulated genes in scramble control tumor cell clusters compared to PERK‐KO clusters ( n = 3). (B) Volcano plot showing PERK‐related metabolites identified through targeted metabolomics ( n = 6). (C) Heatmap showing metabolites involved in the methionine cycle in scramble and PERK‐KO tumor cell clusters ( n = 6). (D) Intracellular metabolites in NC‐oe and PERK‐oe tumor cell clusters ( n = 6). (E) The schematic diagram for the conversion of [ 13 C 5 ]‐methionine into various metabolites (left) and LC‐MS quantification of M+5 methionine, M+5 SAM, and M+4 SAH following a 16 h incubation with [ 13 C 5 ]‐methionine in scramble and PERK‐KO tumor cell clusters (right) ( n = 4). (F) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC cells cultured in CM or medium lacking Ser, Gly, Met, or Cys for 24 h ( n = 5). (G‐I) Western blot analysis of metabolic enzyme expression in MDA‐MB‐231/LM2‐CTC and B16F10 tumor cell clusters with or without PERK (G), ATF4 expression in MDA‐MB‐231/LM2‐CTC with or without PERK (H), and ATF4 and MAT2A expression in MDA‐MB‐231/LM2‐CTC with or without ATF4 (I). (J) Relative MAT2A expression in MDA‐MB‐231/LM2‐CTC transfected with shNC or shATF4 #2 ( n = 3). (K) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC transduced with shNC or shMAT2A #3 and treated with or without SAM for 24 h (left) or pretreated with PF9366 prior to SAM (right) ( n = 3). (L) Schematic of the in vivo experiment design. (M) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). (N) Representative H&E staining images (left) and quantification of pulmonary nodules (right) in NCG mice at 2 weeks after tail vein injection of MDA‐MB‐231/LM2‐CTC clusters (shNC and shMAT2A #3) ( n = 5). (O) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters were cultured in CM or MRM for 24 h before tail vein injection into NCG mice, with lung metastases assessed at 24 h. (P) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). Results represent mean ± SD. Student's t‐test in D, E, J, M, and P; one‐way ANOVA test in F and K. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; FC, fold change; Sc, scramble; PERK, Protein kinase RNA‐like endoplasmic reticulum kinase; PERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; LC‐MS, liquid chromatography‐mass spectrometry; Met, methionine; SAM; S‐adenosylmethionine; SAH, S‐adenosylhomocysteine; CM, control medium; Ser, serine; Gly, glycine; Cys, cysteine; PSAT1, phosphoserine aminotransferase 1; PHGDH, phosphoglycerate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MAT2A, methionine adenosyltransferase 2A; ATF4, activating transcription factor 4; shNC, negative control short hairpin RNA; NC‐oe, negative control‐overexpression; PERK‐oe, PERK‐overexpression; shATF4, short hairpin RNA of ATF4; shMAT2A, short hairpin RNA of MAT2A; MRM, methionine‐restricted medium; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; BLI, lung Bioluminescence imaging; H&E, hematoxylin and eosin; ANOVA, analysis of variance.

Article Snippet: To assess the impact of MAT2A inhibition on S‐adenosylmethionine (SAM) synthesis, MDA‐MB‐231/LM2‐CTC clusters were treated with PF9366 (a MAT2A inhibitor, 10 μmol/L; HY‐107778, MedChemExpress) for 24 h. To evaluate the effect of imatinib on the phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) signaling pathway, MDA‐MB‐231/LM2‐CTC, B16F10, and PDX‐1 cells were treated with imatinib (1 μmol/L.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Incubation, Cell Culture, Western Blot, Expressing, Transfection, Transduction, In Vivo, Imaging, Fluorescence, Injection, Staining, Knock-Out, Liquid Chromatography, Mass Spectrometry, Negative Control, shRNA, Over Expression

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Methionine metabolism promotes H3K4me3 methylation modification to upregulate PDGFB expression in CTC clusters. (A) Western blot analysis of PERK, MAT2A, and H3K4me3 levels in control and PERK‐KO MDA‐MB‐231/LM2‐CTC clusters treated with or without PF9366 or SAM. (B) The volcano plot depicting differentially expressed genes affected by methionine ( n = 3). (C) Metaplot comparing H3K4me3 enrichment profiles in MDA‐MB‐231/LM2‐CTC clusters cultured in CM or MRM. The plot is centered on TSS (±3.0 Kb) ( n = 3). (D) Bioinformatics analysis filtered out 10 survival‐related genes as downstream targets of H3K4me3. (E) Heatmap showing survival‐associated gene expression in MDA‐MB‐231/LM2‐CTC clusters under CM or MRM conditions ( n = 3). (F) Genome browser tracks of H3K4me3 at the PDGFB gene locus by ChIP‐seq ( n = 3). (G) TEM images of MDA‐MB‐231/LM2‐CTC cluster. (H‐J) Representative immunofluorescence images (left) and quantification (right) of PDGFB in CTC clusters from NCG‐MDA‐MB‐231/LM2 mouse model (H, n = 20); or in the complete or dispersed CTC clusters (I, n = 15); or in control or PDGFB‐knockdown CTC clusters (J, n = 15). Arrows indicate cell‐cell border; circle indicates intracellular regions. (K) Representative images of flow cytometry (left) and quantification of AnnexinV + MDA‐MB‐231/LM2‐CTC clusters (right) in control (shNC) and PDGFB‐knockdown (shP#1/2/3) groups ( n = 5). (L) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters (shNC and shPDGFB #3) were injected into NCG mice via the tail vein, and metastatic burden was assessed by bioluminescence imaging at 24 h. (M‐N) Bioluminescence images (left) and quantification of fluorescence intensity (right) showing lung metastasis after tail vein injection at 24 h ( n = 5). Results represent mean ± SD. One‐way ANOVA test in A and K; student's t‐test in H, I, J, M, and N. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; FC, fold change; PERK, PRKR‐like endoplasmic reticulum kinase; ERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; MAT2A, methionine adenosyltransferase 2A; H3K4me3, trimethylation of histone H3 at lysine 4; SAM, S‐adenosylmethionine; CM, control medium; MRM, methionine‐restricted medium; No., number; TSS, transcription start site; ChIP‐seq, chromatin immunoprecipitation sequencing; RNA‐seq, RNA sequencing; PDGFB, platelet‐derived growth factor subunit B; IGF2BP3, insulin‐like growth factor 2 mRNA‐binding protein 3; PHGDH, phosphoglycerate dehydrogenase; ANXA11, annexin A11; ITGA6, integrin subunit alpha 6; ITGB4, integrin subunit beta 4; LOX, lysyl oxidase; PIK3C2B, phosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 beta; IRF9, interferon regulatory factor 9; ATP2A3, ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 3; TEM, transmission electron microscopy; DAPI, 4′,6‐diamidino‐2‐phenylindole; shNC, negative control short hairpin RNA; shP#, short hairpin RNA targeting PDGFB; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; BLI, lung Bioluminescence imaging; ANOVA, analysis of variance.

Article Snippet: To assess the impact of MAT2A inhibition on S‐adenosylmethionine (SAM) synthesis, MDA‐MB‐231/LM2‐CTC clusters were treated with PF9366 (a MAT2A inhibitor, 10 μmol/L; HY‐107778, MedChemExpress) for 24 h. To evaluate the effect of imatinib on the phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) signaling pathway, MDA‐MB‐231/LM2‐CTC, B16F10, and PDX‐1 cells were treated with imatinib (1 μmol/L.

Techniques: Methylation, Modification, Expressing, Western Blot, Control, Cell Culture, Gene Expression, ChIP-sequencing, Immunofluorescence, Knockdown, Flow Cytometry, Injection, Imaging, Fluorescence, Knock-Out, RNA Sequencing, Derivative Assay, Binding Assay, Transmission Assay, Electron Microscopy, Negative Control, shRNA

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Schematic diagram of UPR‐PERK signaling contributing to CTC cluster survival in bloodstream microenvironment. In response to the bloodstream microenvironment, CTC clusters activate the adaptive UPR, which stimulates the PERK‐ATF4‐MAT2A signaling axis to regulate intracellular SAM availability. The accumulation of SAM in CTC clusters induces H3K4me3 modification at the PDGFB promoter, leading to enhanced PDGFB expression. Secreted PDGFB accumulates in the intercellular region of clustered cells, providing shared survival signals to cells within the cluster, thus sustaining CTC cluster survival in the bloodstream. Abbreviations: CTCs, circulating tumor cells; UPR, unfolded protein response; PERK, protein kinase R‐like endoplasmic reticulum kinase; ATF4, activating transcription factor 4; MAT2A, methionine adenosyltransferase 2A; SAM, S‐adenosylmethionine; H3K4me3, histone H3 lysine 4 trimethylation; PDGFB, platelet‐derived growth factor subunit B; PDGFRβ, platelet‐derived growth factor receptor beta.

Article Snippet: To assess the impact of MAT2A inhibition on S‐adenosylmethionine (SAM) synthesis, MDA‐MB‐231/LM2‐CTC clusters were treated with PF9366 (a MAT2A inhibitor, 10 μmol/L; HY‐107778, MedChemExpress) for 24 h. To evaluate the effect of imatinib on the phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) signaling pathway, MDA‐MB‐231/LM2‐CTC, B16F10, and PDX‐1 cells were treated with imatinib (1 μmol/L.

Techniques: Modification, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: A. Immunoblot analysis of MAT2 in MCF-7 and TAMR-MCF-7 cells. Each lane represents different sample. B. Basal MAT2A promoter reporter activities in MCF-7 and TAMR-MCF-7 cells. MCF-7 and TAMR-MCF-7 cells were seeded in 12 wells plate for 1 day. Both the cell types (60% confluency) were then transiently co-transfected with MAT2A-luc reporter plasmid containing −570/+61 bp human MAT2A promoter (1 μg/well) and phRL-SV (hRenilla) (1 ng/well). Dual luciferase reporter assays were performed on the lysed cells 18 h after transfection in serum free condition. Reporter gene activity was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent mean ± SD with 6 different samples (significant versus MCF-7 cells, ** P < 0.01). C. MAT2A mRNA levels in MCF-7 and TAMR-MCF-7 cells. MAT2A mRNA levels were determined by quantitative RT-PCR. Data represent the mean ± SD ( n = 4)(significant versus MCF-7 cells, ** P < 0.01). D. Immunohistochemistry of MAT2A in human breast cancer tissues. Four TAM-responsive and four TAM-resistant cases were estimated. The brown color staining represents MAT2A expression. When we determined immunoreactivity in IgG-incubated breast cancer tissue samples (negative control), we could not detect any positive staining. E. MAT2A immunoblot analyses in T47D cells. The basal MAT2A levels were compared in T47D, MCF-7, TAMR-MCF-7 and MDA-MB-231 cells.

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Incubation, Negative Control

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: A. AP-1 activities in MCF-7 and TAMR-MCF-7 cells. Left; AP-1 minimal reporter activity. MCF-7 and TAMR-MCF-7 cells were co-transfected with pAP-1-Luc reporter (1 μg/well) and phRL-SV plasmids (hRenilla, 1 ng/well). Luciferase reporter activity was determined described as figure legend of Figure . Data represent mean ± SD with 6 different samples (significant versus MCF-7 cells, ** P < 0.01). Right; Nuclear levels of c-Jun, c-Fos, Jun-B and Jun-D in MCF-7 and TAMR-MCF-7 cells. Each AP-1 protein was detected in the nuclear fractions isolated from serum-deprived MCF-7 and TAMR-MCF-7 cells. B. Left; Effect of c-Jun siRNA on MAT2A expression level in TAMR-MCF-7 cells. TAMR-MCF-7 cells were transfected with control or c-Jun siRNA (60 p mole/well) for 36 h. The protein levels of MAT2 and c-Jun in total cell lysates were determined by immunoblotting. Middle; Effect of c-Jun siRNA on MAT2A gene transcription in MCF-7 and TAMR-MCF-7 cells. Both cell lines were co-transfected with MAT2A-luc reporter plasmid containing −570/+61 bp human MAT2A promoter (1 μg/well), phRL-SV (1 ng/well) and control or c-Jun siRNA (20 p mole/well) for 36 h. Luciferase reporter activity was determined described as figure legend of figure . Data represent mean ± SD with 3 different samples. Right; Effect of c-Jun siRNA on AP-1 reporter activity in TAMR-MCF-7 cells. TAMR-MCF-7 cells were co-transfected with control or c-Jun siRNA (20 p mole/well) and pAP-1-Luc (1 μg/well)/phRL-SV (1 ng/well) for 36 h. Data represent mean ± SD with 6 different samples (significant versus control siRNA-transfected TAMR-MCF-7 cells, ** P < 0.01). C. Nrf2/ARE activities in MCF-7 and TAMR-MCF-7 cells. Upper; nuclear level of Nrf2 were detected in the nuclear fractions isolated from serum-deprived MCF-7 and TAMR-MCF-7 cells. Lower; the basal ARE reporter activities in MCF-7 and TAMR-MCF-7 cells. Both cell lines were transiently co-transfected with pGL-ARE-luc plasmid (1 μg/well) and phRL-SV (1 ng/well). Dual luciferase reporter assays were performed on the lysed cells 18 h after transfection in serum free condition. Data represent mean ± SD with 6 different samples (significant versus MCF-7 cells, ** P < 0.01). D. Effect of Nrf2 siRNA on MAT2A expression (upper) and MAT2A gene transcription (lower) in TAMR-MCF-7 cells. Upper; MCF-7 cell and TAMR-MCF-7 cells were transfected with control or Nrf2 siRNA (60 p mole/well) for 36 h. MAT2 and Nrf2 protein expression was determined by immunoblotting. Lower; effect of Nrf2 siRNA on MAT2A gene transcription in MCF-7 and TAMR-MCF-7 cells. Both cell lines were co-transfected with MAT2A-luc reporter plasmid containing −570/+61 bp human MAT2A promoter (1 μg/well), phRL-SV (1 ng/well) and control or Nrf2 siRNA (20 p mole/well) for 36 h. Data represent mean ± SD with 3 different samples.

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Activity Assay, Transfection, Luciferase, Isolation, Expressing, Control, Western Blot, Plasmid Preparation

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: A. NF-κB activation in TAMR-MCF-7 cells. Upper; Nuclear level of p65. p65 protein levels were detected in the nuclear fractions isolated from serum-deprived MCF-7 and TAMR-MCF-7 cells. Lower; NF-κB minimal reporter activity. MCF-7 and TAMR-MCF-7 cells in 12 well plates were co-transfected with NF-κB-luc reporter (1 μg/well) and phRL-SV plasmids (1 ng/well) for 18 h in serum free condition. Luciferase reporter activity was determined described as figure legend of Figure . Data represent mean ± SD with 6 different samples (significant versus MCF-7 cells, ** P < 0.01). B. Effects of TPCK (NF-κB inhibitor) on MAT2A expression (upper) and NF-κB reporter activity (lower) in TAMR-MCF-7 cells. Upper; TAMR-MCF-7 cells were exposed to TPCK with the indicated concentration for 24 h. MAT2 expression level was determined from total cell lysates using immunoblotting. Lower; MCF-7 and TAMR-MCF-7 cells were co-transfected with NF-κB-luc reporter (1 μg/well) and phRL-SV plasmids (1 ng/well). Concomitantly, TPCK with the indicated concentration was treated to the transfected TAMR-MCF-7 cells for 24 h. Luciferase reporter activity was determined described as figure legend of figure . Data represent mean ± SD with 3 different samples (significant versus MCF-7 cells, ** P < 0.01; significant versus vehicle-treated TAMR-MCF-7 cells, ## P < 0.01). C. Effect of TPCK on MAT2A gene transcription. MCF-7 and TAMR-MCF-7 cells were co-transfected with MAT2A-luc reporter plasmid (1 μg/well) and phRL-SV plasmids (1 ng/well). Concomitantly, TPCK with the indicated concentration was treated to the transfected TAMR-MCF-7 cells for 24 h. Data represent mean ± SD with 3 different samples (significant versus MCF-7 cells, ** P < 0.01; significant versus vehicle-treated TAMR-MCF-7 cells, # P < 0.05). D. Effect of IκBα overexpression on the nuclear expression of p65 and MAT2A gene transcription in TAMR-MCF-7 cells. TAMR-MCF-7 cells were co-transfected with MAT2A-luc reporter plasmid (1 μg/well) and pCMV5 or IκBα overexpression plasmid (0.5 μg, respectively), and phRL-SV plasmids (1 ng/well) for 18 h in serum free condition. Data represent mean ± SD with 4 different samples (significant versus pCMV5-transfected MCF-7 cells, ** P < 0.01; significant versus pCMV5-transfected TAMR-MCF-7 cells, # P < 0.05).

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Activation Assay, Isolation, Activity Assay, Transfection, Luciferase, Expressing, Concentration Assay, Western Blot, Plasmid Preparation, Over Expression

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: A. Down-regulation of miR-146a and miR-146b expression in TAMR-MCF-7 cells. miR-146a and miR-146b expression in MCF-7 and TAMR-MCF-7 cells were determined by using miScript PCR kit with miScript primers specific for mature miR-146a and miR-146b. Samples were normalized to small nRNA U6. B. Effects of miR-146a and miR-146b mimics on the miR-146a/b expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells were cultured in 6 wells plate and then transfected with miR-146a or miR-146b mimics for 36 h (120 p mole/well). miR-146a and miR-146b levels were determined by using miScript PCR kit with miScript primers specific for mature miR-146a and miR-146b. Samples were normalized to small nRNA U6. C. Effects of miR-146a/b mimic on NF-κB activity and MAT2A protein expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells were cultured in 6 wells plate and then transfected with miR-146a or miR-146b mimics (120 p mole/well) for 36 h and immunoblottings were performed. The data were confirmed by two independent experiments. D. Effects of miR-146b mimic on MAT2A mRNA expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells were cultured in 6 wells plate and then transfected with miR-146a or miR-146b mimics (120 p mole/well) for 36 h. MAT2A mRNA levels were determined by quantitative RT-PCR. Data represent mean ± SD with 3 different samples (significant versus control mimic miR-treated TAMR-MCF-7 cells, * P < 0.05). E. miR-146b expression in T47D cells. miR-146b expression in MCF-7 and T47D cells were determined by using miScript PCR kit with miScript primers specific for mature miR-146b. Samples were normalized to small nRNA U6.

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Expressing, Cell Culture, Transfection, Activity Assay, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: A. Effect of MAPK inhibitors and PI3K inhibitor on MAT2A expression. Upper; MAT2A protein expression was completely suppressed by 24 h incubation of LY294002 (LY, 20 μM), a PI3K inhibitor in TAMR-MCF-7 cells. PD98059 (PD, 20 μM, ERK inhibitor), SB203580 (SB, 10 μM, p38 kinase inhibitor) and SP600125 (SP, 10 μM, JNK inhibitor) were also used. Lower; Densitometry data. Data represent mean ± SD with 3 different samples (significant versus control MCF-7 cells, ** P < 0.01; significant versus TAMR-MCF-7 cells, ## P < 0.01). B. Effect of Mycp85 (dominant negative mutant form of PI3K) overexpression on MAT2A gene transcription. TAMR-MCF-7 cells were co-transfected with MAT2A-luc reporter plasmid (1 μg/well) phRL-SV plasmids (1 ng/well) and pCMV5 or Mycp85 overexpression plasmid (0.5 μg/well) for 18 h in serum free condition. Luciferase reporter activity was determined described as figure legend of Figure . Data represent mean ± SD with 3 different samples (significant versus pCMV5-transfected MCF-7 cells, ** P < 0.01; significant versus pCMV5-transfected TAMR-MCF-7 cells, ## P < 0.01). C. Effect of PI3K inhibition on NF-κB activity. Left; nuclear p65, nuclear Nrf2, and nuclear c-Jun detection. TAMR-MCF-7 cells were treated with LY294002 (LY, 20 μM) for 24 h and nuclear levels of p65, Nrf2, and c-Jun were detected by immunoblotting. Right; Effect of Mycp85 overexpression on NF-κB minimal reporter activity. TAMR-MCF-7 cells were co-transfected with pNF-κB-Luc reporter plasmid (1 μg/well) and pCMV5 or Mycp85 overexpression plasmid (0.5 μg/well). Then, pNF-κB-Luc reporter activity was determined 18 h after transfection in serum-free condition. Data represent mean ± SD with 3 different samples (significant versus pCMV5-transfected MCF-7 cells, ** P < 0.01; significant versus pCMV5-transfected TAMR-MCF-7 cells, ## P < 0.01). D. Effect of LY294002 (PI3K inhibitor, LY, 20 μM) on miR-146b expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells was exposed to LY294002 for 24 h and then miR-146b level was determined by using miScript PCR. Samples were normalized to small nRNA U6. Data represent mean ± SD with 3 different samples (significant versus vehicle-treated TAMR-MCF-7 cells, ** P < 0.01).

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Expressing, Incubation, Control, Dominant Negative Mutation, Over Expression, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Inhibition, Western Blot

Journal: Oncotarget

Article Title: Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells

doi: 10.18632/oncotarget.5298

Figure Lengend Snippet: The scheme shows positive feedback loop between the PTEN-controlled PI3K/Akt pathway and miR-146b-controlled NF-κB/MAT2A expression.

Article Snippet: Sections were then incubated overnight with anti-MAT2A antibody (NBP1-28605; Novus biological, CO, USA) at 4°C.

Techniques: Expressing

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A The enrollment of TCFA-positive patients defined by OCT and schematic representation of the patients screened in the discovery cohort. This figure was created using images from PowerPoint and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/dmkgnf0 . B Partial least-squares discrimination analysis (PLS-DA) 3D score plots. C Volcano plot of significantly differential metabolites in monocytes. D Detection of monocytes methionine metabolism concentrations ( n = 38 subjects). E The final FCT was determined by averaging three measurements performed at the thinnest part of the fibrous cap. F Vulnerable plaque characteristics between patients with high and low SAM/SAH levels ( n = 38 subjects). G Multivariate logistic regression analysis revealing the relationship between high SAM/SAH levels and TCFA ( n = 76 for total subjects). H Representative OCT images of a normal coronary artery, a vulnerable plaque, and a stable plaque. Scale bar = 5 mm. I Schematic of the methionine metabolism pathway. J The mRNA levels in monocytes isolated from TCFA-positive and TCFA-negative individuals ( n = 38 subjects). UMAP visualization ( K ) and MAT2A expression ( L ) of human atherosclerotic carotid artery cells ( n = 15 patients, GSE253903 ). UMAP visualization ( M ) and MAT2A expression ( N ) of human atherosclerotic carotid artery myeloid cells. Representative immunofluorescence images ( O ) and quantification ( P ) of MAT2A and CD11b in carotid plaques from symptomatic and asymptomatic patients ( n = 6 subjects. Scale bar = 50 µm). C Two-tailed P-values were indicated. D The bar bands defined as 25th and 75th percentiles; the plot top line as median; two-tailed Mann–Whitney P -values are indicated. F The box shows the 25th, 50th and 75th percentiles of the data. The whiskers represent minimum and the maximum; Chi-squared test and two-tailed Mann–Whitney P -values was used. G The error bars represent the 95% CI for the OR, with their centers (red squares) indicating the point estimates; two-tailed P -values are shown. ( J , P ) Data are presented as the mean ± SD; two-tailed P -values are shown. L , N Log normalized using the ‘NormalizeData’ function in Seurat with default parameters. CKD chronic kidney disease, FCT fibrous cap thickness, MAT2A methionine adenosyltransferase Ⅱ alpha, OCT optical coherence tomography, SAH, S-adenosyl-homocysteine, SAM S-adenosyl-methionine, TCFA thin-cap fibroatheroma, UMAP uniform manifold approximation and projection. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Isolation, Expressing, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Tomography

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: Both groups of 8-week-old female and male MAT2A fl/fl ApoE -/- and MAT2A CKO ApoE -/- mice were fed an HFD for 16 weeks. A Schematic figure showing the experimental strategy for the HFD feeding and the subsequent analysis. This figure was created using images from Photoshop and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/3iunh0m . B SAM and SAH concentrations as well as SAM/SAH ratio for mice monocyte cell lysate mass spectrometry samples ( n = 6). C Representative photographs of atherosclerotic plaques in the aortic arches and their branches in the 2 groups ( n = 10 mice per genotype). Representative images and quantification of Oil Red O-stained aortas ( D , n = 10 samples. Scale bar = 2 mm) and aortic roots ( E , n = 10 independent experiments. Scale bar = 200 µm). F , H , E and Masson’s trichrome staining of representative aortic root sections. Scale bar = 200 µm. G Quantification of plaque area, necrotic core, and collagen ( n = 10 samples). H and I , Immunohistochemistry of CD68 + macrophages and vulnerability index in the aortic roots ( n = 10 samples). Scale bar = 100 µm. Data are presented as the mean ± SD and comparisons were made using unpaired Student’s t -test; the two-tailed P -values are shown. α-SMA α-smooth muscle actin, HFD high-fat diet. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Mass Spectrometry, Staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A Bubble chart of GO term enrichment analysis of downregulated DEGs based on RNA-seq in BMDMs of MAT2A CKO compared with MAT2A fl/fl mice. Flow cytometry was performed to quantify CD45 + CD11b + CD115 + Ly6C hi monocytes from bone marrow or peripheral blood ( B , n = 6 independent experiments), MHC Ⅱ and CCR2 ( C , n = 6 samples) on CD45 + CD68 + macrophages from aortic plaques of MAT2A CKO ApoE -/- and MAT2A fl/fl ApoE -/- mice fed a 16-week HFD. D – J Analysis of BMDMs from MAT2A CKO and MAT2A fl/fl mice treated with or without SAM (200 µM) following LPS and IFN-γ stimulation. Representative flow cytometry plots and quantification of CD45 + CD11b + CD86 + macrophages ( D , n = 6 samples), transwell ( E , n = 6 samples, Scale bar = 100 µm), and wound assay ( F , n = 6 samples. Scale bar = 500 µm). The mRNA levels of Il1b , Il6 , Nos2 ( G , n = 6 samples) and Ccl2 , Ccl7 and Cxcl1 ( H , n = 6 samples) measured by qRT-PCR. I , J The THP-1 cells were transfected with siNC or siMAT2A followed by LPS and IFN-γ treatment with or without SAM (200 µM). The mRNA levels of Il1b , Il6 , Nos2, Ccl2 , Ccl7 and Cxcl1 were analyzed by qRT-PCR ( n = 6 samples). A P -values were derived from hypergeometric distribution. Data are presented as the mean ± SD. B , C Unpaired Student’s t -test was used; two-tailed P values are shown. D – J One-way ANOVA was used; the adjusted P -values are shown. 8-week-old female and male mice were both used. BL blood, BM bone marrow, BMDMs bone marrow-derived macrophages, DEGs differentially expressed genes, GO gene ontology, IFN-γ interferon-γ, LPS lipopolysaccharide. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: RNA Sequencing, Flow Cytometry, Quantitative RT-PCR, Transfection, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A Immunoblots of H3K4me3, H3K36me3, and H3K27me3 levels in BMDMs from MAT2A fl/fl and MAT2A CKO mice ( n = 3). B Representative immunofluorescence images and quantification for H3K4me3 and CD68 in aortic root plaque from MAT2A fl/fl ApoE -/- and MAT2A CKO ApoE -/- mice fed a 16-week HFD ( n = 7). Scale bar = 100 µm. C – G Analysis of BMDMs between MAT2A CKO and MAT2A fl/fl mice. C Heatmaps for H3K4me3 binding peaks in BMDMs from MAT2A fl/fl and MAT2A CKO mice. D Venn diagram reflecting overlapping downregulated genes with H3K4me3 modification combining the CUT&Tag and RNA-seq data. Bubble charts of GO ( E ) and KEGG ( F ) analysis according to downregulated genes with decreased H3K4me3 modification. G IGV tracks revealing the results of CUT&Tag reads (H3K4me3 binding) distributions in indicated genes. H , I Analysis of BMDMs between MAT2A CKO and MAT2A fl/fl mice treated with or without SAM (200 µM) in the presence of LPS and IFN-γ. H ChIP-qPCR validation of H3K4me3 enrichment on the promoter regions of Aim2 , Ccl2 , and Mmp9 ( n = 6 samples). I ELISA analysis of AIM2, CCL2, and MMP9 in cellular supernatant ( n = 6 independent experiments). Data are presented as the mean ± SD. A , B Unpaired Student’s t -test was used; two-tailed P values are shown. E P -values were derived from hypergeometric distribution. H and I One-way ANOVA was used; the adjusted P -values are shown. Both 8-week-old female and male mice were used. ChIP-qPCR chromatin immunoprecipitation-qPCR, H3K4me3 histone h3 lysine 4 trimethylation, H3K27me3 histone H3 lysine 27 trimethylation, H3K36me3 histone h3 lysine 36 trimethylation, KEGG kyoto encyclopedia of genes and genomes, TSS translation start site. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Western Blot, Immunofluorescence, Binding Assay, Modification, RNA Sequencing, ChIP-qPCR, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Chromatin Immunoprecipitation

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A – I The HFD-fed ApoE -/- mice were received either FIDAS-5 or vehicle every other day for 6 weeks. A, Flowchart illustrating the experimental procedure. This figure was created using images from Photoshop and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/h76jjnw . B Representative photographs of atherosclerotic plaques in the aortic arches and their branches. Representative images and quantification of Oil Red O-stained aortas ( C , n = 8. Scale bar = 2 mm) and aortic roots ( D and E , n = 8. Scale bar = 200 µm). F Representative images of H&E and Masson’s trichrome-stained aortic roots. Scale bar = 200 µm. G Quantification of plaque area, percentage of necrotic core, and collagen of aortic roots ( n = 8 per group). H , I Immunohistochemistry of CD68 + macrophages and vulnerability index in the aortic root plaque of FIDAS-5 treated mice, n = 8 per group. Scale bar = 100 µm. J – R Representative images of female and male mice fed MRD or CD for 6 weeks after a 10-week HFD. J Schematic figure showing the experimental strategy and subsequent analysis. This figure was created using images from Photoshop and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/h76jjnw . K Representative images of aortic arches and their branches in the indicated group. Representative images and quantification of Oil Red O-stained aortas ( L , n = 8. Scale bar = 2 mm) and aortic roots ( M and N , n = 8. Scale bar = 200 µm). O Representative images of H&E and Masson’s trichrome-stained aortic root. P Quantification of plaque area, percentage of necrotic core, and collagen, n = 8. Scale bar = 200 µm. Q and R Immunohistochemical staining CD68 + macrophages and vulnerability index, n = 8. Scale bar = 100 µm. Data are presented as mean ± SD and comparisons were made using unpaired Student’s t -test; two-tailed P values are shown. 8-week-old female and male ApoE -/- mice were used. FIDAS-5, a MAT2A inhibitor; MRD, methionine-restricted diet. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Staining, Immunohistochemistry, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A Norepinephrine levels in serum from TCFA-positive and TCFA-negative individuals ( n = 38 independent experiments). B Correlation between serum the ratio of SAM/SAH and norepinephrine levels. C The percentage of patients presenting with TCFA in 3 coronary arteries varied according to their levels of the SAM/SAH ratio and norepinephrine. D MAT2A expression in peripheral blood monocytes from 6-OHDA-treated ApoE -/- mice ( n = 3, one representative experiment out of three was shown). E Representative images and quantification of H&E and Oil Red O staining in aortic root treated with 6-OHDA (250 mg/kg) and AAV-MAT2A, n = 6. Scale bar = 100 µm. F Immunoblots of aortic plaques from ApoE -/- mice treated with 6-OHDA ( n = 3). G , Mat2a mRNA expression in BMDMs pre-treated with 20 nM RAPA followed by 10 mM norepinephrine ( n = 6, one representative experiment out of three was shown). H Transcript levels of c-Myc and Mat2a in BMDMs transfected with the indicated siRNAs ( n = 6). I Occupancy analysis of c-MYC by ChIP-qPCR in BMDMs treated with DMSO vehicle or RAPA (20 nM) ( n = 6). A Two-tailed Mann–Whitney P -values are indicated; Data are shown as median with IQR. B Pearson correlation coefficient test was used; the regression coefficients and two-tailed P -values are shown. C Chi-squared test was used; Data are shown as %. D – F Unpaired Student’s t -test was used; the two-tailed P -values are shown; Data are presented as mean ± SD. G , I Two-way ANOVA was used; The adjusted P -values are shown; Data are presented as mean ± SD. H One-way ANOVA was used; The adjusted P -values are shown. 8-week-old female and male ApoE -/- mice were used. Data are presented as mean ± SD. AAV adeno-associated virus, RAPA rapamycin, 6-OHDA 6-Hydroxydopamine. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Expressing, Staining, Western Blot, Transfection, ChIP-qPCR, Two Tailed Test, MANN-WHITNEY, Virus

Journal: Nature Communications

Article Title: MAT2A promotes atherosclerotic plaque vulnerability by mediating epigenetic reprogramming of macrophages

doi: 10.1038/s41467-025-66121-z

Figure Lengend Snippet: A Study design in the validation cohort. This figure was created using images from PowerPoint and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/qbkjhvs . Detection of monocytes SAM and SAH concentrations ( B ), and SAM/SAH ratio ( C ) of TCFA-positive and TCFA-negative individuals ( n = 100 subjects). D Serum norepinephrine levels from TCFA-positive and TCFA-negative individuals ( n = 100 independent experiments). Qualitative and quantitative OCT analysis of vulnerable plaque characteristics between patients with high and low SAM/SAH levels ( E ) or with high and low norepinephrine levels ( F ) ( n = 100 independent experiments). Pearson correlation analysis of monocytes SAM/SAH ratio ( G ) or serum norepinephrine levels ( H ) with the thinnest FCT and mean lipid arc. I Two-tailed Pearson’s linear regression analysis between the SAM/SAH ratio and norepinephrine levels. J Multivariate logistic regression analysis depicting the relationship between the SAM/SAH ratio, norepinephrine levels, and TCFA ( n = 200 for total subjects). K Receiver operating characteristic curve of SAM/SAH, norepinephrine, and combined both for TCFA. L HR for incident 5-year MACE based on multivariable Cox proportional hazards regression analysis. Adjusted for age, gender, traditional coronary risk factors, and statin at discharge ( n = 200 for total subjects). M A Kaplan-Meier survival curve plots the 5-year MACE-free survival among 4 subgroups. N MAT2A-mediated monocyte methionine metabolism is closely associated with the presence of TCFA in patients. MAT2A, which is induced by the norepinephrine–mTOR–c-MYC axis, is critical for endowing monocytes/macrophages with proinflammatory state and migratory capacity during the development of atherosclerosis through H3K4me3 modification. Myeloid-specific MAT2A ablation, pharmacological blockade with FIDAS-5, or a low-methionine diet attenuate monocyte/macrophage inflammation and migration, thereby reducing atherosclerotic progression and plaque vulnerability. This figure was created using images from PowerPoint and BioRender. Created in BioRender. wan, p. (2025) https://BioRender.com/gylosq5 . B – D Two-tailed Mann–Whitney P -values are indicated. E , F The box shows the 25th, 50th and 75th percentiles of the data. The whiskers represent minimum and the maximum. Data are presented as % or median with IQR, Chi-squared test or Mann-Whitney test was used, and two-tailed P -values were calculated. G – I Pearson correlation coefficient test was used; the regression coefficients and two-tailed P -values are shown. J Data points indicate OR, and 95% confidence intervals are represented by line length; two-tailed P -values are shown. K The 95% confidence interval is shown between brackets. L Data points indicate HR and 95% CI were represented by line length. M P -values were calculated with log rank test. HR hazard ratio. Source data are provided as a Source Data file.

Article Snippet: To establish a pharmacological MAT2A inhibition model , , ApoE -/- mice were administered the MAT2A inhibitor FIDAS-5 (10 mg/kg, HY-136144, MedChemExpress, New Jersey, USA) by intragastric gavage.

Techniques: Biomarker Discovery, Two Tailed Test, Modification, Migration, MANN-WHITNEY