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: Nature Communications

Article Title: Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells

doi: 10.1038/s41467-018-06406-8

Figure Lengend Snippet: Loss of PKM2 impairs methylation capacity, reduces DNA methylation and leads to the expression of endogenous retroviral elements. a Western blot analysis of MAT2A, P53, P21 and PKM2 in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs. b Relative levels of S-adenosylmethionine and S-adenosylhomocysteine and SAM/SAH ratio in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 3). c Percentage 5-methylcytosine (5mC) levels in control and PKM2 KD ECs ( n = 4). d Heat map of protein expression for components of the cellular response to viral infection in control and PKM2 KD ECs. e Representative confocal projections of total double-stranded RNA (dsRNA) staining in control and PKM2 KD ECs (scale bar = 20 μm). f Normalized fluorescence intensity for dsRNA staining in control and PKM2 KD ECs ( n = 7). g Relative mRNA expression of the indicated endogenous retroviruses in control and PKM2 KD ECs ( n = 3). h Restriction digestion of bisulfite-treated DNA amplified from the MLT1B and MER4D genomic loci from control and PKM2 KD ECs (U, undigested/unmethylated DNA; D, digested/methylated DNA). i Relative mRNA expression of the indicated endogenous retroviruses and interferon-stimulated genes in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs. b , c , f , g , i Data represent means ± s.d. (*** P < 0.001, ** P < 0.01, * P < 0.05 by one-way analysis of variance (ANOVA) followed by Tukey’s HSD test)

Article Snippet: The following antibodies were used: PKM1 (Cell Signaling Technology, #7067, 1:1000), PKM2 (Cell Signaling Technology, #4053, 1:1000), P21 (Cell Signaling Technology, #2947, 1:1000), P53 (Cell Signaling Technology, #9282, 1:500), P53 (Santa Cruz, sc-126, 1:1000), RELB (Cell Signaling Technology, #10544, 1:1000), MAT2A (Novus Biologicals, #NB110-94158, 1:2000), β-actin (Cell Signaling Technology, #8457, 1:1000), GAPDH (Cell Signaling Technology, #2118, 1:2000) and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2004, 1:10,000).

Techniques: Methylation, DNA Methylation Assay, Expressing, Retroviral, Western Blot, Control, Infection, Staining, Fluorescence, Amplification

Journal: Cancers

Article Title: MAT2A as Key Regulator and Therapeutic Target in MLL r Leukemogenesis

doi: 10.3390/cancers12051342

Figure Lengend Snippet: Human CRISPR/Cas9- MLL r model reveals MAT2A as a possible target in MLL r leukemia. ( A ) RNA sequencing of human CRISPR/Cas9- MLL-AF4 (two different donors, n = 2) compared to the respective controls (ctrl, n = 2, CD34 + huCB cells nucleofected with Cas9 alone and cultured for the same time) revealed upregulated expression of MAT2A . Values in normalized reads per kilobase million (nRPKM), each dot represents a sample. ( B ) Validation of MAT2A expression was performed by RT-qPCR. MLL-AF4 and MLL-AF9 cells were normalized to culture-expanded CD34 + huCB control cells (ctrl). Experiment was performed in biological duplicates ( n = 2) with horizontal bars representing the mean. Error bars indicate standard deviation (SD). One-way ANOVA was used with Dunnett correction: * p < 0.05. ( C ) Representative Western blot analysis shows increased MAT2A expression in MLL r cells compared to culture expanded CD34 + huCB control cells (factor 1.8 for MLL-AF4 and factor 1.7 for MLL-AF9 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. Blots are cropped and full images are displayed in . ( D ) Upregulated MAT2A expression in leukemic patient samples compared to other cancer types (data from oncomine.org) . Boxes indicate the range from the 25th through 75th percentiles; the horizontal lines represent the median; error bars indicate the range from 10th through 90th percentiles; the dots show the maximum and minimum values. Student’s t test was used: * p < 0.05. ( E ) MAT2A expression in MLL r leukemia compared to non- MLL r leukemia and healthy controls, respectively (GSE28497, data from oncomine.org) . Boxes indicate the range from 25th through 75th percentiles; the horizontal lines represent the median; error bars indicate the range from 10th through 90th percentiles; the dots show the maximum and minimum values. Mann–Whitney U test was used: * p < 0.05.

Article Snippet: MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [ , , , ].

Techniques: CRISPR, RNA Sequencing, Cell Culture, Expressing, Biomarker Discovery, Quantitative RT-PCR, Control, Standard Deviation, Western Blot, MANN-WHITNEY

Journal: Cancers

Article Title: MAT2A as Key Regulator and Therapeutic Target in MLL r Leukemogenesis

doi: 10.3390/cancers12051342

Figure Lengend Snippet: MAT2A inhibition impairs the proliferation and viability of MLL r cells, resulting in reduced SAM levels and histone methylation. ( A ) MLL-AF4/-AF9 or culture-expanded CD34 + huCB control cells were treated with increasing concentrations of PF-9366 or vehicle (DMSO) for 6 days and the relative cell count was determined by flow cytometry using counting beads. Pooled data of three biological replicates ( n = 3) performed in technical triplicates are shown. IC 50 values of the dose–response curves were interpolated from a four-parameter logistic model constrained to 0 and 1 in GraphPad Prism. Dots represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. ( B ) Proliferation curves were assessed by treating the indicated cells with PF-9366 (10, 15 µM) or control (DMSO) and counting cells following Trypan blue staining every second day. The mean of pooled data of three biological replicates ( n = 3) performed in technical triplicates is shown. Dots represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. ( C ) Cell viability of MLL r and control cells upon inhibition treatment with PF-9366 (10, 15 µM) or control (DMSO) was determined after 6 days by fluorescence-based read of alamarBlue assay. Experiment was performed in three biological replicates ( n = 3) and technical triplicates. Bars represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. ( D ) MLL r cells were treated with 15 µM PF-9366 or DMSO as a control for 6 days. Intracellular SAM levels of 10 4 cells were quantified by fluorescence-based Bridge-It Assay Platform Technology using a SAM standard curve. Results of two independent experiments ( n = 2) are displayed. Student’s t -test was used: *, p < 0.05. ( E ) Western blot analyses of extracted histones from MLL r cells treated with 15 µM or DMSO as the control for 6 days are displayed showing decreased global methylation of H3K4me3, H3K79me1, H3K79me2, and H4R3me2. Blots are cropped and full images are displayed in .

Article Snippet: MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [ , , , ].

Techniques: Inhibition, Methylation, Control, Cell Counting, Flow Cytometry, Staining, Fluorescence, Alamar Blue Assay, Western Blot

Journal: Cancers

Article Title: MAT2A as Key Regulator and Therapeutic Target in MLL r Leukemogenesis

doi: 10.3390/cancers12051342

Figure Lengend Snippet: Inhibition of MAT2A reduces DNA synthesis and induces apoptosis of MLL r cells. MLL- AF4/-AF9 or culture-expanded CD34 + huCB control cells were treated with PF-9366 (10 or 15 µM) or vehicle (DMSO) for 6 days. ( A ) Pooled data (left) and representative (right) cell cycle analyses of single cells from three biological replicates ( n = 3) performed in technical triplicates are shown. Data were acquired using BrdU staining and flow cytometry. Bars represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. ( B ) Annexin V staining revealed translocation of phosphatidylserine to the outer leaflet of the plasma membrane in apoptotic cells. Pooled (left) and representative (right) data from three biological replicates ( n = 3) performed in technical triplicates are shown. Dots represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05.

Article Snippet: MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [ , , , ].

Techniques: Inhibition, DNA Synthesis, Control, BrdU Staining, Flow Cytometry, Staining, Translocation Assay, Clinical Proteomics, Membrane

Journal: Cancers

Article Title: MAT2A as Key Regulator and Therapeutic Target in MLL r Leukemogenesis

doi: 10.3390/cancers12051342

Figure Lengend Snippet: MAT2A inhibition increases differentiation and diminishes MLL r-specific target gene expression. MLL-AF4/-AF9 or culture-expanded CD34 + huCB control cells were treated with 10 or 15 µM of PF-93 66 or vehicle (DMSO) for 6 days. ( A ) Representative morphologies of MLL r and control cells are shown displaying increased cell differentiation and cell death after drug treatment. Scale bars define 20 µm. ( B ) Pooled (left) and representative (right) CD14 expression of living single cells from three biological replicates ( n = 3) performed in technical triplicates measured by flow cytometry are shown. Bars represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. ( C ) Target gene expression was analyzed by RT-qPCR demonstrating decreasing levels of MEIS1 and HOXA9 upon inhibition with PF-9366 (10 or 15 µM). Experiment was performed in three biological replicates ( n = 3) and technical triplicates. Bars represent the mean. Error bars indicate SD. One-way ANOVA was used with Dunnett correction: *, p < 0.05. Student’s t test was used for MLL-AF9 : *, p < 0.05.

Article Snippet: MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [ , , , ].

Techniques: Inhibition, Targeted Gene Expression, Control, Cell Differentiation, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Cancers

Article Title: MAT2A as Key Regulator and Therapeutic Target in MLL r Leukemogenesis

doi: 10.3390/cancers12051342

Figure Lengend Snippet: Downstream effects of MAT2A inhibition on gene expression. ( A ) MLL-AF4 cells were treated with 15 µM of PF-9366 or vehicle (DMSO) for 4 days and two biological replicates ( n = 2) were used for RNA-seq. DEGs are displayed with 38 genes upregulated and 36 genes downregulated. The interactome of all DEGs is shown in ( B ) with the main effect of MAT2A inhibition on cell cycle progression notable by the interacting genes CDKN1A/p21 , CDK1 , RB1 , and MDM2. ( C ) For long-term experiments, MLL-AF4/-AF9 cells were treated with 10 or 15 µM of PF-9366 or vehicle (DMSO) for a minimum of 2 weeks. Stable inhibition of proliferation is shown by two biological replicates ( n = 2) performed in technical triplicates.

Article Snippet: MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [ , , , ].

Techniques: Inhibition, Gene Expression, RNA Sequencing

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 2: SAMe and MTA induce the expression of miR-34a and miR-34b and all four treatments induce apoptosis and inhibit growth of colon cancer cells. (A) RKO (left) and SW620 (right) cells were treated with 250 μM SAMe or MTA for 24 hours. The mRNA levels of miR-34a and miR-34b were compared to vehicle treated controls using real-time PCR. Results represent mean ± SEM from 4-5 experiments, *p<0.001 vs. control. (B) MAT2A 3’UTR reporter activity in RKO and SW620 cells treated with miR-34a, miR-34b or empty vector (EV). Results represent mean ± SEM from 3 experiments, *p<0.01 vs. EV. RKO (C) and SW620 (D) cells were treated with 250 μM SAMe or MTA, overexpression of miR-34a or miR-34b as described in Methods for 24 hours and were processed for apoptosis, growth by BrdU, miR-34 and miR-34b transfection efficiency measurements. Results represent mean ± SEM from 3 experiments, *p<0.01 vs. EV.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Activity Assay, Plasmid Preparation, Over Expression, Transfection

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 3: Effects of SAMe, MTA, miR-34a and miR-34b on MAT2A and MAT2B expression. RKO (A) and SW620 (B) cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A mRNA levels, MAT2A and MAT2B protein levels (MATα2 and MATβ) were measured by real-time PCR and Western blotting, respectively. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 to 4 experiments, *p<0.05 (real-time PCR) and *p<0.01 (Western blotting) vs. empty vector (EV) control. (C) RKO cells were treated with 10 nM siRNA against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression was measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.001 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 4: Effects of MAT2A, MAT2B, miR-34a and miR-34b in colon cancer cell migration, invasion and growth. RKO cells were transfected with empty vector (EV), miR-34a, miR-34b, MAT2A or MAT2B expression vectors alone or in combination for 24 hours for measurement of cell migration (A), invasion (B) and growth (C) as described in Methods. Results represent mean ± SEM from 3 to 4 experiments, *p<0.05 vs. EV. †p<0.05 vs. MAT2A overexpression. (D) shows transfection efficiency. (E) RKO cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.04 vs. EV.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Migration, Transfection, Plasmid Preparation, Expressing, Over Expression, Western Blot, Control

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 6: MAT2A and MAT2B expression is upregulated in human prostate and pancreatic cancers. (A) Representative IHC of MATα2 and MATβ is shown from normal (n = 8) and prostate cancer patient tissues (n = 40). (B) IHC of MATα2 and MATβ from 4 separate normal pancreatic tissues and PDACs are shown, scale = 100 μm. Magnification x 20.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 8: MAT2A and MAT2B expression is down-regulated by SAMe, MTA, miR-34a and miR-34b in prostate cancer cell line. CWR22Rv1 cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A and MAT2B mRNA (A) and protein (B) levels were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 to 4 experiments, *p<0.04 vs. empty vector (EV) control. (C) Effect of SAMe and MTA treatment (250 μM for 24 hours) on miR-34a and miR-34b expression in CWR22Rv-1 cells. Results represent mean ± SEM from 5 experiments, *p<0.05 vs. control. (D) CWR22Rv-1 cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.02 vs. EV. (E) CWR22Rv-1 cells were transfected with EV, MAT2A or MAT2B overexpression vector and cell migration was measured as described in Methods. Results represent mean ± SEM from 3 experiments, *p<0.002 vs. EV control.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control, Transfection, Migration

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 9: MAT2A and MAT2B expression is down-regulated by SAMe, MTA, miR-34a and miR-34b in pancreatic cancer cell line. MIA PaCa-2 cells were treated with 250 μM SAMe or MTA, or overexpression of miR-34a or miR-34b as described in Methods for 24 hours. MAT2A and MAT2B mRNA (A) and protein (B) levels were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.02 vs. empty vector (EV) control. (C) Effect of SAMe and MTA treatment (250 μM for 24 hours) on miR-34a and miR-34b expression in MIA PaCa-2 cells. Results represent mean ± SEM from 4 experiments, *p<0.05 vs. control. (D) MIA PaCa-2 cells were transfected with MAT2A overexpression vector in DDK tag for 24 hours and Western blotting was done for MATα2, MATβ, p-ERK, total ERK and Actin for housekeeping control. Densitometric values are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.05 vs. EV. (E) MIA PaCa- 2 cells were transfected with EV, MAT2A or MAT2B overexpression vector and cell migration was measured as described in Methods. Results represent mean ± SEM from 3 experiments, *p<0.03 vs. EV control.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control, Transfection, Migration

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 10: MAT2A and MAT2B proteins stabilize each other in CWR22Rv-1 and MIA PaCa-2 cells. CWR22Rv-1 (A) and MIA PaCa-2 (B) cells were treated with 10 nM siRNA against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.03 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 11: MAT2A and MAT2B proteins stabilize each other in RKO, CWR22Rv-1 and MIA PaCa-2 cells. RKO (A), CWR22Rv-1 (B) and MIA PaCa-2 (C) cells were treated with a second siRNA (10 nM) against MAT2A or MAT2B, or scramble siRNA control (Sc) for 48 hours and MAT2A and MAT2B expression were measured by real-time PCR and Western blotting. Densitometric changes are summarized below the blots. Results represent mean ± SEM from 3 experiments, *p<0.05 vs. Sc.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

doi: 10.18632/oncotarget.20234

Figure Lengend Snippet: Figure 12: Summary diagram of miR-34a/b-MAT2A/MAT2B axis and treatment with SAMe or MTA. In normal non- hepatic tissues, miR-34a and miR-34b negatively regulate MAT2A expression mainly by suppressing its protein translation. In cancer cells miRNA-34a and miR-34b are often down-regulated, releasing the inhibition on MAT2A expression. This indirectly raises MAT2B expression as the two proteins interact and stabilize each other. Higher MAT2A and MAT2B expression leads to cancer cell growth, migration and invasion. Treatment with SAMe or MTA in cancer cells increases the expression of miR-34a and miR-34b. Potential mechanisms involved include via lowering DNA methylation of miR-34a/b promoters, and inhibition of IL-6/STAT3 signaling pathway. SAMe and MTA could also suppress MAT2A and MAT2B by other mechanisms not shown. Collectively this results in lower MAT2A and MAT2B expression, increased apoptosis, decreased cell growth, migration and metastasis.

Article Snippet: MAT2A 3’UTR reporter assay MAT2A 3’UTR construct was purchased from Origene (Rockville, MD).

Techniques: Expressing, Inhibition, Migration, DNA Methylation 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