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: 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