Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 enhances host m6A modification and promotes MAT2A expression. ( A ), ( B ) Caco2 cells were infected with SARS-CoV-2 at a MOI of 2. ( A ) Total RNA was extracted at 24, 48, and 72 h post-infection (hpi) and analyzed by dot blot to examine m6A modification. Methylene blue staining was used as a loading control.( B ) Expression levels of m6A machinery proteins in Caco-2 cells infected with SARS-CoV-2. ( C ) MeRIP-seq analysis identified m6A peaks in the SARS-CoV-2 genome isolated from Caco-2 cells infected with SARS-CoV-2 at a MOI of 4. ( D ) Relative abundance of SARS-CoV-2 RNA in 1 µg of total RNA from MeRIP-seq samples. ( E ) Schematic representation illustrating the relationship between MAT2A and m6A modification. ( F ) Protein levels of MAT2A and MAT2B in Caco-2 cells infected with SARS-CoV-2. The α2 and α2’ are two isoforms of the MAT2A protein. ( G ) MAT2A was knocked down in 293 T cells using short hairpin RNA (shRNA), with a non-targeted shRNA (shNC) serving as the control. ( H ), ( I ) The shNC or shMAT2A#1 293 T cell lines were cultured in DMEM containing 2% FBS for 24 h. ( H ) Levels of m6A modification in the indicated 293 T cells. ( I ) Concentrations of methionine and S-adenosylmethionine (SAM) were quantified by UPLC-MS/MS in the indicated cell lines. Data are presented as mean ± SEM of three independent experiments. Statistical significance was determined, with * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Infection, Dot Blot, Staining, Control, Isolation, shRNA, Cell Culture, Tandem Mass Spectroscopy

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 increases host m6A modification by inducing MAT2A expression. ( A ) Expression of MAT2A in 293 T cells transfected with varying doses of HA-nsp14 plasmid. ( B ) Immunoblots of 293 T cells transfected with plasmids encoding either wild-type nsp14, the H268A mutant, or the D331/G333A mutant. ( C ) Dot blot analysis of m6A modification in 293 T cells transfected with increasing doses of HA-nsp14 plasmid. ( D ) MeRIP-RT-qPCR analysis showing the levels of m6A in HA-nsp14 mRNA. Fold enrichment of m6A was normalized to the IgG control. ( E ), ( F ) Quantification of m6A modification in 293 T cells transfected with plasmids encoding either wild-type nsp14 or the D331/G333A mutant. ( G ) Comparative DNA sequence analysis of plasmids encoding wild-type HA-nsp14 and the D331/G333A mutant. ( H ) RT-qPCR analysis of mRNA abundance in the experiment described in panel f. The grayscale intensity was measured using Image J software. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Dot Blot, Quantitative RT-PCR, Control, Sequencing, Software

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 upregulates MAT2A expression by activating the mTORC1 signalling pathway. (A) Immunoblots of Caco2 cells infected with SARS-CoV-2 at a MOI of 2, analyzed at 24, 48, and 72 hpi. (B) Immunoblots of 293 T transfected with HA-nsp14 plasmid in a dose-dependent manner. (C) Transcript levels of MAT2A in 293 T cells transfected with indicated plasmids. (D) Immunoblots of 293 T cells grown overnight in the absence of serum, then treated with either vehicle (H2O) or insulin (500 nM, 30 min), or 293 T cells transfected with HA-nsp14 and treated with either vehicle (DMSO) or rapamycin (20 nM). (E – F) RAPTOR was knocked down in 293 T cells using a shRNA. (E) Immunoblots of the indicated 293 T cells transfected with HA-nsp14 in a dosedependent manner. (F) Quantification of m6A modification in the indicated 293 T cells by dot blot analysis. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with *P < 0.05, **P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, shRNA, Modification, Dot Blot

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: β-Coronaviruses stimulate SAM synthesis through the mTORC1 pathway to facilitate viral replication. ( A ) Caco2 cells were infected with HCoV-OC43, and total RNA was measured for m6A modification by dot blot analysis. ( B ) Immunoblot of Caco2 cells infected with HCoV-OC43. ( C ) Immunoblot of 293 T cells transfected with plasmids encoding SARS-CoV-1, SARS-CoV-2, and HCoV-OC43 nsp14. ZIKV-NS5 (an N7-MTase) was used as a negative control. ( D ) Immunoblot of MAT2A knockdown Caco2 cells infected with HCoV-OC43. ( E ) – ( H ) Caco2 and HRT-18 cells were cultured in the absence of methionine (Met-free). ( E ) Immunoblot of Caco2 cells infected with HCoV-OC43 with L-methionine (L-Met) supplemented in a dose-dependent manner. ( F ) Immunoblot of HRT-18 cells treated as in ( E ). ( G ) Immunoblot of Caco2 cells infected with HCoV-OC43 with SAM supplemented in a dose-dependent manner. ( H ) Immunoblot of HRT-18 cells treated as in ( G ). Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Infection, Modification, Dot Blot, Western Blot, Transfection, Negative Control, Knockdown, Cell Culture

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: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: PPRE regions in the rat MAT2A promoter.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques:

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. Control and RSG-treated BSC cells were transfected with wild type MAT2A promoter (W) and its PPRE mutants designated as M1, M2, M4, M5, M6 and the triple PPRE mutant M1/2/4. Mutated sites in each PPRE are indicated in bold. The luciferase activity in all samples was normalized to that of pGL3-Basic and expressed as a percentage of the wild type promoter activity in control cells. Results represent mean ± S.E. from four experiments in duplicates, *p<0.005, **p<0.05 vs. wild type MAT2A promoter in control cells. B. PPRE deletion mutants were transfected into control and RSG-treated BSC cells. Normalized luciferase activity was expressed as a percentage of the b2A promoter in control cells. Results represent mean ± S.E. from four experiments, *p<0.005, **p<0.05 vs. b2A construct in control cells.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Transfection, Mutagenesis, Luciferase, Activity Assay, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: BSC cells or primary rat HSCs were treated with RSG or DMSO (control) for 48 hours as described under “Experimental procedures”. A. Total RNA from BSC cells was subjected to real-time RT-PCR analysis and expression of MAT2A and PPARγ was compared to that of control. Results represent mean ± S.E. from three experiments in duplicates, *p<0.005 vs. control. B. Total cellular protein from BSC cells was subjected to western blotting for detection of MAT2A or PPARγ. Representative images and densitometric analysis (mean ± S.E.) from three experiments in duplicates is shown. *P<0.005, **p<0.05 vs. control. C. Luciferase activity from RSG-treated BSC cells transfected with the MAT2A promoter construct was normalized to that of pGL3-Basic and expressed as a fold of control cells. Results represent mean ± S.E. from four experiments in triplicates, *p<0.005 vs. control. D. Luciferase activity from RSG-treated primary rat HSCs transfected with the MAT2A promoter construct was normalized to that of pGL3-Basic and expressed as a fold of control cells. Results represent mean ± S.E. from three HSC preparations in duplicates, *p<0.005 vs. control.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. Control and RSG-treated BSC cells were subjected to ChIP to measure the binding of PPARγ to the MAT2A PPRE sites as described under “Experimental procedures”. Input genomic DNA (Input) was used as the loading control and immunoprecipitation with a GFP antibody was used as a negative control. Results are representative agarose gel images of three experiments in duplicates. B. Genomic DNA from immunoprecipitated chromatin as in ‘A’ was quantitated by ChIP real-time PCR as described under “Experimental procedures”. The differential target site occupancy of PPARγ on MAT2A was normalized to input genomic DNA and expressed as fold over control. Results represent mean ± S.E. from three experiments in duplicates. *P<0.005, **P<0.05 vs. control. C. The binding of PPARγ to MAT2A PPREs from BDL HSCs and sham controls was assessed as described in ‘A’. Representative agarose gel images from five HSC preparations in duplicates are shown. D. Genomic DNA from immunoprecipitated chromatin described in ‘C’ was quantitated by ChIP real-time PCR as described under “Experimental procedures”. The differential target site occupancy of PPARγ on MAT2A was normalized to input genomic DNA and expressed as fold over sham. Results represent mean ± S.E. from five HSC preparations in duplicates. *P<0.005 vs. sham.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. RSG-treated BSC cells were transfected with PPARγ siRNA or a negative control siRNA as described under “Experimental procedures”. By real-time RT-PCR, the expression of MAT2A and PPARγ in knockdown cells was compared to that of negative control siRNA. Results represent mean ± S.E. from three experiments in duplicates, *p<0.005 vs. negative control siRNA. B. Cells were treated as in ‘A’ and total cellular protein was subjected to western blotting with antibodies against MAT2A or PPARγ. Representative images and densitometric analysis (mean ± S.E.) from three experiments in duplicates is shown. **P<0.05 vs. negative control siRNA. C. PPARγ knockdown was performed in RSG-treated BSC cells followed by transfection with the MAT2A promoter or pGL3-Basic vector. The luciferase activity in PPARγ knockdown cells was normalized to that of pGL3-basic and expressed as a fold of negative control siRNA. Results represent mean ± S.E. from three experiments in triplicates, **p<0.05 vs. negative control siRNA.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

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

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. BSC cells were transduced with PPARγ Adv or negative control Adv as described under “Experimental procedures”. By real-time RT-PCR, the expression of MAT2A and PPARγ mRNA after PPARγ Adv transduction was compared to that of negative control Adv. Results represent mean ± S.E. from four experiments in duplicates, *p<0.005, **p<0.05 vs. negative control Adv. B. Cells were treated as in ‘A’ and total cellular protein was subjected to western blotting with antibodies against MAT2A or PPARγ. Representative images and densitometric analysis (mean ± S.E.) from three experiments in duplicates is shown. **P<0.05 vs. negative control Adv. C. BSC cells transduced with PPARγ Adv or negative control Adv for 72 hours were transfected with the MAT2A promoter construct or the pGL3-basic vector during the last 24 hours of transduction. The luciferase activity in PPARγ Adv-transduced cells was normalized to that of pGL3-Basic and expressed as a fold of negative control Adv. Results represent mean ± S.E. from five experiments in triplicates, **p<0.05, vs. negative control Adv.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Transduction, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. Chromatin extracted from HSCs isolated from BDL livers and sham control was immunoprecipitated with a PPARβ antibody as described under “Experimental procedures”. Input genomic DNA (Input) was used as the loading control and immunoprecipitation with an antibody against GFP was used as a negative control. Representative agarose gel images from three HSC preparations in duplicates are shown. *P<0.005, **p<0.05 vs. sham. B. Genomic DNA from immunoprecipitated chromatin described in ‘A’ was quantitated by ChIP real-time PCR as described under “Experimental procedures”. The differential target site occupancy of PPARβ on MAT2A was normalized to input genomic DNA and expressed as fold over control. Results represent mean ± S.E. from three HSC preparations in duplicates. *P<0.005, **p<0.05 vs. sham. C. BSC cells (left panel) or culture-activated primary rat HSCs (right panel) were transfected with a PPARβ siRNA or a negative control siRNA as described under “Experimental procedures”. By real-time RT-PCR, the expression of MAT2A and PPARβ in knockdown cells was compared to that of negative control siRNA. Results represent mean ± S.E. from five experiments in duplicates (BSC cells) or four HSC preparations in duplicates. *p<0.005 vs. negative control siRNA. D. Cells were treated as in ‘C’ and total cellular protein was subjected to western blotting with antibodies against MAT2A or PPARβ. Representative images and densitometric analysis (mean ± S.E.) from four to five experiments of BSC cells (left panel) or four HSC preparations (right panel) in duplicates is shown. *P<0.005, **p<0.05 vs. negative control siRNA. E. PPARβ knockdown was performed in BSC cells followed by transfection with the rat MAT2A promoter or pGL3-basic as described under “Experimental procedures”. The luciferase activity in PPARβ knockdown cells was normalized to that of pGL3-Basic and expressed as a fold over negative control siRNA. Results represent mean ± S.E. from four experiments in triplicates, **p<0.05 vs. negative control siRNA.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Isolation, Immunoprecipitation, Negative Control, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: TRANSCRIPTIONAL REGULATION OF METHIONINE ADENOSYLTRANSFERASE 2A BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN RAT HEPATIC STELLATE CELLS

doi: 10.1002/hep.25594

Figure Lengend Snippet: A. A pCMV6-rMAT2A construct was transfected into BSC cells treated with RSG as described in “Experimental procedures”. Total protein was subjected to western blotting with antibodies against MAT2A, PPARγ, PPARβ, C/EBPβ or α-SMA. Representative images and densitometric analysis (mean ± S.E.) from four experiments in duplicates is shown. *P<0.005, **p<0.05 vs. empty vector. B. RSG-treated BSC cells were transfected as described in ‘A’ and total RNA was assessed by real-time PCR for the expression of PPARγ and α-SMA. Results represent mean ± S.E. from four experiments in duplicates. *P<0.005, **p<0.05 vs. empty vector.

Article Snippet: Total cellular protein was subjected to western blotting using antibodies for MAT2A (Novus Biologicals, Littleton, CO), PPARγ, PPARβ, C/EBPβ (Santa Cruz Biotechology, Santa Cruz, CA), and control, β-actin (abcam).

Techniques: Construct, Transfection, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Role of methionine adenosyltransferase 2A in bovine preimplantation development and its associated genomic regions

doi: 10.1038/s41598-017-04003-1

Figure Lengend Snippet: Immunofluorescence analysis of MAT2A protein in bovine oocytes and preimplantation embryos. Confocal images that transverse at least one nucleus or pronucleus are shown. ( A ) Immature oocyte, ( B ) mature oocyte, ( C ) 1-cell, ( D ) 2-cell, ( E ) 8-cell, ( F ) morula, ( G ) blastocyst, ( H ) hatched blastocyst, and ( I ) negative control in which the primary antibody was omitted from the immunofluorescence of a hatched blastocyst sample. Scale bars represent 50 µm. ( A’ – I’ ) Nuclear counterstaining with propidium iodide. ( J ) and ( K ) Immunoblotting of 1-cell embryo (n = 120) and blastocyst (n = 120) lysates using the monoclonal MAT2A antibody used in the immunofluorescence and ChIP-seq analyses. Hep G2 Cell Lysate (50 µg protein) was used as a positive control.

Article Snippet: A mouse monoclonal anti-MAT2A antibody (clone AT3A2; ab86424, Abcam) was diluted 250 times with blocking solution and co-incubated with samples for 12 h at 4 °C.

Techniques: Immunofluorescence, Negative Control, Western Blot, ChIP-sequencing, Positive Control

Journal: Scientific Reports

Article Title: Role of methionine adenosyltransferase 2A in bovine preimplantation development and its associated genomic regions

doi: 10.1038/s41598-017-04003-1

Figure Lengend Snippet: Data generated by ChIP-seq analysis of bovine blastocyst using MAT2A antibody.

Article Snippet: A mouse monoclonal anti-MAT2A antibody (clone AT3A2; ab86424, Abcam) was diluted 250 times with blocking solution and co-incubated with samples for 12 h at 4 °C.

Techniques: Generated, Control

Journal: Scientific Reports

Article Title: Role of methionine adenosyltransferase 2A in bovine preimplantation development and its associated genomic regions

doi: 10.1038/s41598-017-04003-1

Figure Lengend Snippet: Characterization of MAT2A-ChIP specific peaks. ( A ) Chromosome distribution of 76 MAT2A-ChIP specific peaks. The figure was generated using the web-based CEAS ( http://liulab.dfci.harvard.edu/CEAS/ ) tool. ( B ) Distribution of the peak-associated genes in terms of where the central point of the peak is located among different genomic regions. The bovine genome was divided into the following four categories: upstream (<100 kb) of a transcription start site (TSS), downstream (<100 kb) of a transcription end site (TES), the intron, and intergenic (>100 kb from a TSS or TES) regions. The numbers indicate those of genes for the upstream, intron, and downstream regions and those of peaks for the intergenic regions, respectively. ( C ) ChIP-qPCR validation of several MAT2A-associated peaks. Relative enrichments to IgG control are shown as the mean value of two to five ChIP-qPCR analyses for each peak. The peaks are shown with ID number (Table ) and their associated genes in parentheses. The GAPDH promoter was used as a negative control for enrichment.

Article Snippet: A mouse monoclonal anti-MAT2A antibody (clone AT3A2; ab86424, Abcam) was diluted 250 times with blocking solution and co-incubated with samples for 12 h at 4 °C.

Techniques: Generated, ChIP-qPCR, Biomarker Discovery, Control, Negative Control

Journal: Scientific Reports

Article Title: Role of methionine adenosyltransferase 2A in bovine preimplantation development and its associated genomic regions

doi: 10.1038/s41598-017-04003-1

Figure Lengend Snippet: Gene ontology (GO) annotation in terms of biological process enriched from 35 genes associated with the MAT2A-ChIP specific peaks. All significant (P < 0.05) secondary level terms obtained as output from a web-based GO analysis toolkit, AgriGO ( http://bioinfo.cau.edu.cn/agriGO/ ), are shown. Blue columns show the percentage of genes among the peak-associated genes, and green columns show the percentage of genes within the whole genome.

Article Snippet: A mouse monoclonal anti-MAT2A antibody (clone AT3A2; ab86424, Abcam) was diluted 250 times with blocking solution and co-incubated with samples for 12 h at 4 °C.

Techniques:

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A Overall levels of m 6 A in the hippocampi of the AAV-control ( n = 3) and AAV-sh-METTL16 group ( n = 3) were tested by m 6 A colorimetry. B Overall levels of m 6 A in the HT22 cells of the Lenti-control group ( n = 4) and Lenti-sh-METTL16-3 group ( n = 3) were tested by m 6 A colorimetry. C Expression level of MAT2A mRNA was tested by qRT–PCR in the hippocampi of MWM-trained ( n = 6) and untrained mice ( n = 6). D , E Representative images ( D ) and quantification of MAT2A ( E ) in the hippocampi of MWM-trained ( n = 5) and untrained mice ( n = 5) tested by western blotting. F , G Representative IHC images ( F ) and quantification of MAT2A ( G ) in the hippocampi of MWM-trained ( n = 3) and untrained mice ( n = 3); Scale bars = 500 μm. H Expression level of MAT2A mRNA was tested by qRT–PCR in the hippocampi of the AAV-control group ( n = 3) and AAV-sh-METTL16 group mice ( n = 3). I , J Representative images ( I ) and quantification of MAT2A ( J ) in the hippocampi of the AAV-control group ( n = 5) and AAV-sh-METTL16 group mice ( n = 5) tested by western blotting. K , L Representative IHC images ( K ) and quantification of MAT2A ( L ) in the hippocampi of the AAV-control group ( n = 5) and AAV-sh-METTL16 group mice ( n = 5); Scale bars = 500 μm. M Expression level of MAT2A mRNA was tested by qRT–PCR in the HT22 cells of the Lenti-control group ( n = 3) and Lenti-sh-METTL16-3 group ( n = 3). N , O Representative images ( N ) and quantification of MAT2A ( O ) in the HT22 cells of the Lenti-control group ( n = 4) and Lenti-sh-METTL16-3 group ( n = 4) tested by western blotting. P Representative image of stereotactic injections of AAV-sh-M16+OE-MAT2A in the hippocampi; Scale bars = 500 μm. Q , R Representative images ( Q ) and quantification of MAT2A ( R ) in the hippocampi of the AAV-sh-M16+ctrl group ( n = 5) and AAV-sh-M16+OE-MAT2A group mice ( n = 5) tested by western blotting. S , T Representative IHC images ( S ) and quantification of MAT2A ( T ) in the hippocampi of the AAV-sh-M16+ctrl group ( n = 5) and AAV-sh-M16+OE-MAT2A group mice ( n = 5); Scale bars = 500 μm. U Overexpression efficiency of MAT2A in the hippocampi was detected by qRT–PCR ( n = 4). V Overall levels of m 6 A in the hippocampi of the AAV-sh-M16+ctrl group ( n = 3) and AAV-sh-M16+OE-MAT2A group mice ( n = 3) tested by m 6 A colorimetry. W , X Representative images ( W ) and quantification of MAT2A ( X ) in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 4) tested by western blotting. Y Overexpression efficiency of MAT2A in the HT22 cells was detected by qRT–PCR. Z Overall levels of m 6 A in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 3) were tested by m 6 A colorimetry. Data shown as the mean ± SEM. P -values were determined by two-tailed t -test. * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Control, Colorimetric Assay, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A Representative Golgi-Cox impregnated spines in the hippocampi of the AAV-sh-M16+ctrl and AAV-sh-M16+OE-MAT2A group; Scale bars = 10 μm. B , C Quantification of the spine density ( B ) and the percentage of tin spine, filopodia spine, mushroom-like spine, and stubby spine ( C ) in the hippocampi of the two groups of mice ( n = 36 neurons from 6 mice per group). D Quantification of the spine density of mushroom-like and stubby spine in the hippocampi of the two groups of mice ( n = 36 neurons from 6 mice per group). E , F Representative images ( E ) and quantification of Drebrin, PSD95, and Syp ( F ) in the hippocampi of the two groups of mice were tested by western blotting ( n = 5). G , H Representative IHC images ( G ) and quantification of Drebrin, PSD95, and Syp ( H ) in the hippocampi of the two groups of mice ( n = 5); Scale bars = 500 μm. I , J Representative images ( I ) and quantification of Drebrin, PSD95, and Syp ( J ) in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 4) tested by western blotting. K – N Entries in new arm ( K ), time in new arm ( L ), latency 1st entrance to new arm ( M ), and total distance ( N ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were tested in Y-maze test. O – Q Discrimination index (DI) for new objects of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) was tested in 2 h ( P ) and 24 h ( Q ) after excluding the mice’s preference for object location ( O ) in the ORT experiment. R , S Average velocity ( R ) and latency to find the platform ( S ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the visible platform phase of MWM. T Latency to find the platform of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the training trials phase of MWM. U , V Number of platform crossings ( U ) and time in target quadrants ( V ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the probe trial phase of MWM. Data shown as the mean ± SEM. P- values were determined by two-tailed t -test ( B – D , F , H , J – S , U , V ), and two-way repeated-measures ANOVA ( T ). * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Western Blot, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A , B HT22 cells were treated with 5 μg/ml ActD for up to 8 h with or without prior knockdown of METTL16. The expression levels of MAT2A mRNA ( A ) and U1 snRNA ( B ) were measured on the 0, 2, 4, 6, and 8 h after the ActD addition ( n = 3). C , D RIP-qRT–PCR was employed to investigate the direct interaction of METTL16 protein and MAT2A mRNA in the HT22 cells. Immunoblot analysis of METTL16 was performed as a control ( C ). MAT2A mRNA was assessed by qRT–PCR in endogenous METTL16, or IgG (negative control) immunoprecipitates from HT22 cells and are shown as percentages of input RNA ( n = 3) ( D ). E – G Anti-m 6 A RIP-qRT–PCR using total RNA from HT22 cells with or without METTL16 knockdown. Immunoblot analysis of m 6 A was performed as a control ( E ). Cartoon render of the PCR products in MAT2A mRNA ( F ). The indicated transcripts of MAT2A mRNA ( F ), β-actin, and U1 snRNA were quantified by qRT–PCR and are shown as percentages of input RNA ( G ) ( n = 3). H , I HT22 cells were treated with 5 μg/ml ActD for up to 8 h with prior overexpression of MAT2A mRNA-3′UTR-wild or MAT2A mRNA-3′UTR-mut. The expression levels of MAT2A mRNA ( H ) and U1 snRNA ( I ) were measured on the 0, 2, 4, 6, and 8 h after the ActD addition ( n = 3). Data shown as the mean ± SEM. P -values were determined by two-tailed t -test ( D , G ) and two-way repeated-measures ANOVA ( A , B , H , I ). * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Over Expression, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: The primers used in qRT–PCR experiments.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques:

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: The sequence of wild and mutant-MAT2A mRNA-3′UTR.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Sequencing, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: MATα2 is secreted by CRC cells in extracellular vesicles. A Exopred and Exocarta software predicts MATα2 is secreted via exosomes ( B ) CRC cells transfected to overexpress DDK-MAT2A (M2A) secreted more MATα2 when compared to empty vector (EV). C NanoSight analysis of extracellular vesicles isolated from culture media of CRC cells transfected to overexpress MAT2A (MAT2A OE) and empty vector (EVec) with peak corresponding to size range of exosomes. D Images from NanoSight analysis of extracellular vesicles. E Immunofluorescence microscopy of human hepatocytes treated with exosomes isolated from culture media from CRC transfected to overexpress DDK-MAT2A and empty vector (EVec) shows internalization of EV-MATα2 with localizing to the nuclues via DAPI staining

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Software, Transfection, Plasmid Preparation, Isolation, Immunofluorescence, Microscopy, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 is internalized by hepatocytes and alters MAT1A and MAT2A expression A mRNA levels of MAT1A and MAT2A isolated from human hepatocytes treated with exosomes from CRC cells. B Western blot analysis of endogenous nuclear and cytosolic MATα2 in human hepatocytes treated with exosomes from CRC cells. Lamin B1 and tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Mean ± SEM from n = 3, * p < 0.05 vs. EVec exo. C-D Confocal microscopy of human liver spheroids treated with exosomes from CRC cells transfected with empty vector (EVec) or MAT2A-His vector at day 7 for 24 h showing effect on MATα1 and MATα2-His expression. E mRNA levels of MAT1A and MAT2A isolated from human liver spheroids after the exosome treatment. Mean ± SEM from n = 3, * p < 0.02 vs.control and † p < 0.05 vs. EVec exo

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Expressing, Isolation, Western Blot, Confocal Microscopy, Transfection, Plasmid Preparation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Integrated analysis of MATα2 genomic binding profiles in CRC cells. A Heatmap showing the read density distribution of MATα2 ChIP-seq peaks across all human chromosomes. Read intensities are color-coded from low (purple) to high (yellow) density. B Genomic annotation of MATα2 binding peaks. C Top five DNA-binding motifs enriched within MATα2-bound peaks ranked by motif enrichment score. D Pathway enrichment analysis of MATα2-associated genes. E Predicted consensus sequences logos representing the top three de novo motifs identified from MATα2 binding sites by Jaspar software. F ChIP-seq tracks showing MATα2 (blue), RNA polymerase II (POL II; red) and input control (black) signals across the MAT1A (lower) or MAT2A (upper) locus. The Y-axis represents normalized read enrichment. The bottom track shows the gene structure with exons (black boxes) and introns (dashed lines)

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Binding Assay, ChIP-sequencing, Software, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 acts as a transcription factor to alter MAT1A and MAT2A expression. A Promoter activities in human hepatocytes transfected with human MAT1A or MAT2A promoter constructs and then treated with exosomes from RKO cells expressing empty vector (EVec exo) or MAT2A (EV-MATα2) as described in Methods. B ChIP analysis of the human MAT1A and ( C ) human MAT2A promoters showing binding of MATα2-His to different regions of the promoters. Mean ± SEM from n = 8, * p < 0.05 and ** p < 0.01 vs. EVec exo for MAT1A promoter; n = 7, * p < 0.04 vs. EVec exo for MAT2A promoter

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: EV-MATα2 induces MAT2A expression and oncogenic activity in RKO cells. A RKO cells were treated with exosomes from RKO cells expressing empty vector (EVec exo) or MAT2A-His-Tag vector (EV-MATα2) as described in Methods and cell entry was visualized under fluorescent microscopy using His-tag antibody. B Real-time PCR shows the effect at the MAT2A mRNA level. Mean ± SEM from n = 3, * p < 0.02 vs. EVec exo. C Western blotting was done in total cell lysate, cytoplasmic and nuclear fractions showing increased MATα2 levels. Densitometry were measured by ImageJ. Mean ± SEM from n = 3, * p < 0.002 for total lysate, * p < 0.02 for cytoplasmic, * p < 0.03 for nuclear fractions vs. EVec exo. D ChIP analysis of the human MAT2A promoter showing binding of MATα2-His to different predicted motifs. Mean ± SEM from n = 3, * p < 0.05 vs. EVec exo. Effects of the same treatments on EdU ( E ) ( n = 3, * p < 0.05 and ** p < 0.01 vs. control), migration ( F ) ( n = 3, * p < 0.004 and ** p < 0.0001 vs. 0 h EVec exo, † p < 0.003 vs. 24 h EV-MATα2), and invasion ( G ) ( n = 3, * p < 0.008 vs. EVec exo)

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Expressing, Activity Assay, Plasmid Preparation, Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Control, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: MAT expression in adjacent hepatocytes is altered in human CRLM and patients with CRC secrete two forms of MATα2. A , B Human tissue microarray including normal human liver (NHL, n = 3) and CRLM ( n = 32) were examined using IHC (x100) for MATα1, MATα2, and hepatocyte specific antigen (HSA). Boxed areas are magnified, MATα1 and MATα2 staining in hepatocytes was analyzed by Image J and summarized in the graphs. Mean ± SEM, * p < 0.005 for MATα1, * p < 0.04 for MATα2 vs. NHL. C Human plasma from 5 healthy controls (HC) and 10 CRC patients were western blotted for MATα2 to detect full-length MATα2 (MATα2-fl) and truncated MATα2 (MATα2-t), with RKO lysate as input control. Mean ± SEM. * p < 0.05 vs. HC, ** p < 0.001 vs. HC

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Expressing, Microarray, Staining, Clinical Proteomics, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Cancer cells secrete more truncated MATα2, which is required for survival. A Medium from RKO cells overexpressing MAT2A-His or empty vector (EVec) was separated into exosomes and EV-free media that only has truncated MATα2 (MATα2-t). Note MATα2-His has higher MW than full length endogenous MATα2, which has the same MW as MATα2-t-His. Endogenous MATα2-t has the lowest MW. B MATα2 protein sequence and predicted cleavage site based on PrediSI is at proline 30. C RKO (CRC), MiaPACA (pancreatic adenocarcinoma) and RV1 (prostate adenocarcinoma) cells secrete more MATα2-t as compared to the respective non-malignant cells (HCoEpC, HPDE, RWPE1). Mean ± SEM from n = 3, * p < 0.01 vs. HCoEpC cells; * p < 0.03 and † p < 0.01 vs. HPDE cells; * p < 0.03 and † p < 0.001 vs. RWPE1 cells. D RKO and HT29 cells treated with anti-MATα2 (20 µg/ml) for 48 h and TUNEL staining shows CRC cells underwent apoptosis. E MTT assay in RKO and HT29 cells treated with anti-MATα2 shows a fall in viability. Mean ± SEM from n = 3, * p < 0.03 and † p < 0.04 vs. control. F RKO cells were transfected with MATα2-DDK for 48 h and increasing amount of anti- MATα2 Ab was added, followed by pull-down of MATα2 Ab using beads, then western blotted the pull-down with anti-DDK Ab. The MATα2 antibody was able to bring down freely secreted MATα2 in a dose-dependent manner. This correlated with a dose-dependent increase in active caspase 3

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Plasmid Preparation, Sequencing, TUNEL Assay, Staining, MTT Assay, Control, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A novel role of secreted methionine adenosyltransferase α2 in colorectal liver metastases

doi: 10.1186/s13046-025-03599-x

Figure Lengend Snippet: Secreted MATα2-t activates FAK and is required to maintain MAT2A expression. A RKO cells were treated with EV-free media containing MATα2-t as described in Methods and western blotted for pFAK and total FAK. Mean ± SEM from n = 3, * p < 0.04 vs. EVec ( B ) RKO cells were treated with anti-MATα2 Ab for 48 h and western blotted for pFAK, total FAK, pro-caspase 3 and active caspase 3. Mean ± SEM from n = 3, * p < 0.03 vs. control. C FAK and MAT2A mRNA levels in RKO cells from the above treatments were measured by real-time PCR. Mean ± SEM from n = 3, * p < 0.002 vs. EVec; * p < 0.002 vs. control. D RKO cells were CRISPR/Cas9 gene edited (HDR) to mutate proline to leucine at position 30 (canonical motif: PDLD) and at positions 131 and 133 glycine to leucine (non-canonical motif: GXGD); western blotted for pFAK and FAK. Mean ± SEM from n = 5–6, * p < 0.01 vs. wild-type (WT) for PDLD. E Immunoblotting of secreated MATα2 (MATα2-t) in culture media from RKO cells gene edited PDLD motif. Mean ± SEM from n = 3, * p < 0.04 vs. WT. F PDLD gene edited RKO and HT29 cells exhibited increased apoptosis on TUNEL staining (blue for RKO and brown for HT29 depending to pH culture media)

Article Snippet: The slides were deparaffinized, hydrated, and stained for MATα1 (cat. H0004143-M01; Abnova, Taipei City, Taiwan) MATα2 (cat. NB110-94158; Novus, St. Charles, MI), and hepatocyte-specific antigen using extended antigen retrieval (antigen unmasking solution, cat. H-3301-250; Vector Laboratories, Burlingame, CA) as we described [ ].

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, CRISPR, TUNEL Assay, Staining