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: 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