Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: Primers for real-time PCR.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: Expression of MTA1 in various tissues and organs of adult mice and during embryonic lung development. ( A, B ) MTA1 protein and mRNA expression by Western blotting and real-time PCR, respectively, in different tissues of adult wild-type mice. ( C ) immunohistochemical analysis demonstrates MTA1 expression in the lungs at E16.5, E18.5, and P1. (D) MTA1 protein expression in the lungs at E16.5, E18.5, and P1. Scale bar, 50 μm. ( D, E ) A gradual, but significant decrease in the protein and mRNA levels of MTA1 was observed in the lungs from E16.5 to P1. Actin controls in A and D were blotted in parallel gels with the same amount of loading. n=6, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

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

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: MTA1 deficiency increases infant mortality and impairs growth. ( A ) Postnatal mortality shows that over one-third of homozygous MTA1 -KO mice died within 7 days after birth compared to wild-type mice. ( B, C ) MTA1-deficient mice that did survive exhibited retarded growth and markedly decreased body weight compared to wild-type mice. n=30, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: MTA1 -KO mice present cardiac hypertrophy. ( A ) Representative images of HE-stained MTA1 -KO and wild-type cardiac structures. Compared with wild-type animals, cardiomyocytes of MTA1 -KO mice were bigger and hypertrophied ( p <0.05). ( B ) The HW/BW ratio was significantly higher in MTA1 -KO than in wild-type mice at 8 weeks of age ( p <0.05), indicating MTA1 deficiency induced myocardial hypertrophy in adult mice. ( C, D ) The size of right ventricular cardiomyocytes was significantly larger in MTA1 -KO than in wild-type mice at 8 weeks of age (n=10), * P <0.05, ** P <0.01, *** P <0.001. ( E ) α-SMA immunostaining highlights pulmonary artery wall thickening and hypertension in MTA1 -KO mice (n=6). Scale bar, 50 μm.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

Techniques: Staining, Immunostaining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: MTA1 -KO mice display reduced vascularization of the lungs. ( A–C ) HE and Heme staining demonstrate a significantly reduced number of pulmonary capillaries and red blood cells in the lungs of MTA1 -KO mice compared to wild-type animals. n=10, Scale bar, 50 μm, * P <0.05, ** P <0.01, *** P <0.001. ( D ) Transmission electron micrograph showing a similar decrease in the number of pulmonary capillaries (▽) and red blood cells (RBC, ☆) at E18.5 and 2M in MTA1 -KO mice. n=6, Scale bar, 5 μm.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

Techniques: Staining, Transmission Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: MTA1 deficiency impairs the formation of pulmonary capillary beds. ( A, B ) Immunohistochemistry results showed fewer CD34-positive cells in the lungs of MTA1 -KO animals compared to wild-type mice at E18.5 and 2M. n=10, Scale bar, 50 μm, * P <0.05, ** P <0.01. ( C ) The percentages of ERG positive cells were similar in the alveolar walls of wild-type and MTA 1-KO mice at E18.5 but were increased significantly in the MTA1-deficient mice of 2M. n=10, Scale bar, 50 μm. ( D ) Western blot analysis highlights the downregulation of CD34 in the lungs of MTA1 -KO mice at E18.5 and 2M compared to wild-type animals. ERG levels were similar at E18.5 but increased significantly in the MTA1-deficient mice of 2M. ( E, F ) The same expression tendencies of CD34 and ERG at mRNA levels were confirmed at the E18.5 and 2M stage by real-time PCR analysis. n=10, * P <0.05.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

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

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: Regulation of HIF-1α expression by MTA1. ( A, B ) Real-time PCR and Western blot analysis results indicated that MTA1 correlates well with many lung development-related factors such as VEGF, AQP5, SPB, and β-catenin. ( C, D ) Western blot and real-time PCR showed significant upregulation of HIF-1α and VEGF in 293T and MLE-12 cells overexpressing MTA1 36 h after transfection. n=3, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

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

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice

doi: 10.12659/MSM.905992

Figure Lengend Snippet: A correlation between the expression of MTA1 and HIF-1α in the lungs. MTA1 is an active angiogenic regulator that deacetylates HIF-1α. The MTA1- HIF-1α axis feed into VEGF expression and the formation of pulmonary capillaries.

Article Snippet: Sources of antibodies were as follows: MTA1 (D40D1) XP ® rabbit mAb (#5647, Cell Signaling Technology); CD31 (PECAM-1) (D8V9E) XP ® rabbit mAb (#77699, Cell Signaling Technology); rabbit anti-CD34 (BA0532, Boster Bio-Technology Co., LTD); rabbit anti-VEGF (BA0407, Boster Bio-Technology Co., LTD); ERG (A7L1G) Rabbit mAb (#97249, Cell Signaling Technology); rabbit anti-HIF-1α/HIF1A (PB0245, Boster Bio-Technology Co., LTD); rabbit anti-alpha smooth muscle actin (α-SMA) (ab5694, Abcam); and β-actin (Abcam).

Techniques: Expressing

Journal:

Article Title:

doi:

Figure Lengend Snippet: A. The HEK293T whole cell extracts were used for co-IP with MTA1 or PARP1 anti antibody, and probed for endogenous CASZ1 by western blot (left panel); The mouse ES cell extracts were used for co-IP with MTA1 or PARP1 anti antibody, and probed for endogenous CASZ1 by western blot (right panel). B. Mouse ES cell extracts were fractionated through a gel filtration column, and selected fractions were then resolved by SDS-PAGE and western blotting performed to indentify the indicated proteins.

Article Snippet: For western blot analysis, anti-FLAG M2 monoclonal antibody (Sigma), anti-Gal4 antibody, anti-GAPDH antibody (Santa Cruz Biotechnology), anti-HDAC1, HDAC2, PARP1, XRCC1, XRCC5, XRCC6, RPA, RBAP46/48, CHD4, PAR, MBD3 antibody (Cell Signaling), anti-MTA1, MTA2, MTA3 antibody (Bethyl), were used as primary antibody.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Filtration, SDS Page

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: lncRNA PCAT1 might coordinate ZNF217 to promote CRC adhesion and invasion through regulating MTA2/MTA3/Snai1/E-cadherin signaling.

doi: 10.14715/cmb/2021.67.4.1

Figure Lengend Snippet: Figure 5. The protein expressions of MTA2, MTA3, SNAI1 and E-cadherin in CRC tissue and cell lines. (a) The upregulation of MTA2, MTA3, and SNAI1, and downregulation of E-cadherin in stage 0-I and stage II-III CRC tissues samples. (b) The upregulation of MTA2, MTA3, and SNAI1, and downregulation of E-cadherin in both HT29 and HCT8 cells. * p < 0 05, * p < 0 01 and ***p < 0 001 vs paracancerous CRC or FHC cell line. # p < 0 01, ## p < 0 01, and ### p < 0 001 vs stage 0-I CRC or HT29 cell line.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with Rabbit polyclonal MTA2 (1:3000, 17554-1-AP, Proteintech), MTA3 (1:1000, 14682-1-AP, Proteintech), SNAI1 (1:5000, 20874-1- AP, Proteintech), and mouse anti-human E-cadherin (1:2000, 60335-1-Ig, Proteintech) and beta-actin as internal reference (1:8000, 60008-1-Ig, Proteintech).

Techniques:

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: lncRNA PCAT1 might coordinate ZNF217 to promote CRC adhesion and invasion through regulating MTA2/MTA3/Snai1/E-cadherin signaling.

doi: 10.14715/cmb/2021.67.4.1

Figure Lengend Snippet: Figure 6. ZNF217 knockdown reduces MTA2, MTA3, and E-cadherin proteins expressions, and increases SNAI1 expression in both HT29 (a) and HCT8 (b) cells. * p < 0 05, * p < 0 01 and ***p < 0 001 vs si-NC.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with Rabbit polyclonal MTA2 (1:3000, 17554-1-AP, Proteintech), MTA3 (1:1000, 14682-1-AP, Proteintech), SNAI1 (1:5000, 20874-1- AP, Proteintech), and mouse anti-human E-cadherin (1:2000, 60335-1-Ig, Proteintech) and beta-actin as internal reference (1:8000, 60008-1-Ig, Proteintech).

Techniques: Knockdown, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Leptin-Upregulated Metastasis-Associated Protein 1 Promotes Vasculogenic Mimicry in Breast Cancer Cells

doi: 10.3390/ijms26125726

Figure Lengend Snippet: Leptin upregulates MTA1 expression in human breast cancer cells. MTA1 mRNA expression was analyzed using qPCR in MDA-MB-231 ( A ) and Hs 578T cells ( B ) after leptin treatment for 24 h. Results are expressed as mean ± SD; * p < 0.05 and ** p < 0.01 vs. untreated control. MTA1 protein expression was assessed using Western blot in MDA-MB-231 ( C ) and Hs 578T cells ( D ) after 24 h of leptin treatment.

Article Snippet: MDA-MB-231 cells (1.5 × 10 5 ) and Hs 578T cells (2.0 × 10 5 ) were cultured under optimal conditions and transfected with 1 μg of the control or 0.5 μg of the MTA1 CRISPR activation plasmid (Santa Cruz, Danvers, MA, USA) for 48 h. Transfection was performed using the UltraCruz transfection reagent (Santa Cruz), according to the manufacturer’s protocol.

Techniques: Expressing, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Leptin-Upregulated Metastasis-Associated Protein 1 Promotes Vasculogenic Mimicry in Breast Cancer Cells

doi: 10.3390/ijms26125726

Figure Lengend Snippet: Leptin upregulates MTA1 expression via the Ob-R/STAT3 pathway in human breast cancer cells. MTA1 protein expression was evaluated using Western blot in MDA-MB-231 ( A , C ) and Hs 578T cells ( B , D ) after 24 h of leptin treatment in the presence or absence of Ob-R BP ( A , B ) or AG490 ( C , D ).

Article Snippet: MDA-MB-231 cells (1.5 × 10 5 ) and Hs 578T cells (2.0 × 10 5 ) were cultured under optimal conditions and transfected with 1 μg of the control or 0.5 μg of the MTA1 CRISPR activation plasmid (Santa Cruz, Danvers, MA, USA) for 48 h. Transfection was performed using the UltraCruz transfection reagent (Santa Cruz), according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Leptin-Upregulated Metastasis-Associated Protein 1 Promotes Vasculogenic Mimicry in Breast Cancer Cells

doi: 10.3390/ijms26125726

Figure Lengend Snippet: MTA1 overexpression promotes VM in human breast cancer cells. MDA-MB-231 ( A , C , E ) and Hs 578T cells ( B , D , F ) were transfected with the MTA1 CRISPR activation plasmid for 48 h. MTA1 protein expression was assessed using Western blot in MDA-MB-231 ( A ) and Hs 578T cells ( B ). VM was performed using a 3D culture assay for 16 h in MDA-MB-231 ( C ) and Hs 578T cells ( D ) (40× magnification; scale bar = 500 μm). Results are expressed as mean ± SD; *** p < 0.001 vs. control plasmid. Expression of VM-related proteins was evaluated using Western blot in MDA-MB-231 ( E ) and Hs 578T cells ( F ).

Article Snippet: MDA-MB-231 cells (1.5 × 10 5 ) and Hs 578T cells (2.0 × 10 5 ) were cultured under optimal conditions and transfected with 1 μg of the control or 0.5 μg of the MTA1 CRISPR activation plasmid (Santa Cruz, Danvers, MA, USA) for 48 h. Transfection was performed using the UltraCruz transfection reagent (Santa Cruz), according to the manufacturer’s protocol.

Techniques: Over Expression, Transfection, CRISPR, Activation Assay, Plasmid Preparation, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Leptin-Upregulated Metastasis-Associated Protein 1 Promotes Vasculogenic Mimicry in Breast Cancer Cells

doi: 10.3390/ijms26125726

Figure Lengend Snippet: MTA1 silencing inhibits leptin-induced VM in human breast cancer cells. MDA-MB-231 ( A , C , E ) and Hs 578T cells ( B , D , F ) were treated with leptin 48 h after transfection with MTA1 siRNA. MTA1 protein expression was evaluated using Western blot in MDA-MB-231 ( A ) and Hs 578T cells ( B ). VM was performed using a 3D culture assay for 16 h in MDA-MB-231 ( C ) and Hs 578T cells ( D ) (40× magnification; scale bar = 250 μm). Results are expressed as mean ± SD; *** p < 0.001 vs. untreated control; ### p < 0.001 vs. control siRNA. Expression of VM-related proteins was evaluated using Western blot in MDA-MB-231 ( E ) and Hs 578T cells ( F ).

Article Snippet: MDA-MB-231 cells (1.5 × 10 5 ) and Hs 578T cells (2.0 × 10 5 ) were cultured under optimal conditions and transfected with 1 μg of the control or 0.5 μg of the MTA1 CRISPR activation plasmid (Santa Cruz, Danvers, MA, USA) for 48 h. Transfection was performed using the UltraCruz transfection reagent (Santa Cruz), according to the manufacturer’s protocol.

Techniques: Transfection, Expressing, Western Blot, Control

Journal: International journal of oncology

Article Title: Metastasis-associated protein 1 promotes tumor invasion by downregulation of E-cadherin.

doi: 10.3892/ijo.2014.2253

Figure Lengend Snippet: Figure 2. MTA1 suppression of the E-cadherin promoter is dependent on Snail, Slug, and HDAC1. (A) Knock-down or overexpression of MTA1 influenced E-cadherin expression levels, as assessed by qRT-PCR. (B) In EC-9706 cells, adenovirus expressing MTA1 (Ad-MTA1) inhibited E-cadherin promoter activity in a dose-dependent manner. (C) Mixtures of Ad-MTA1, E-cadherin reporter constructs and siRNAs were transfected into EC-9706 cells for 1 day. At the end of the culture period, cells were lysed and luciferase activity was measured. (D) Chromatin from EJ cells was harvested after the different siRNA treatments for 1 day and then precipitated with antibodies. PCR products from the ChIP assays were visualized by electrophoresis. *P<0.05; MOI, multiplicity of infection; IP, immunoprecipitation.

Article Snippet: The MTA1 siRNA (SC35981), HDAC1 siRNA (SC44208), and Slug siRNA (SC38393) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Quantitative RT-PCR, Activity Assay, Construct, Transfection, Luciferase, Electrophoresis, Infection, Immunoprecipitation

Journal: International journal of oncology

Article Title: Metastasis-associated protein 1 promotes tumor invasion by downregulation of E-cadherin.

doi: 10.3892/ijo.2014.2253

Figure Lengend Snippet: Figure 3. MTA inhibits E-cadherin expression via the E-box sequences. (A) Schematic representation of the E-cadherin reporter constructs. (B) Mutation of the E-box sequences influenced the suppressive effects of MTA1 on the promoter activity of E-cadherin. *P<0.05.

Article Snippet: The MTA1 siRNA (SC35981), HDAC1 siRNA (SC44208), and Slug siRNA (SC38393) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Construct, Mutagenesis, Activity Assay

Journal: International journal of oncology

Article Title: Metastasis-associated protein 1 promotes tumor invasion by downregulation of E-cadherin.

doi: 10.3892/ijo.2014.2253

Figure Lengend Snippet: Figure 4. Binding of MTA1 to the E-cadherin core promoter bearing the two E-box sequences. (A) Upon interaction with the nuclear extract of EC-9706 cells, the probe containing the wild-type E-box sequences gener ated three specific bands (bands a, b, and c), which were competed with by increasing concentrations of the unlabeled oligo. Probes containing muta tions of the E1-box or E3-box sequences gave rise to only one specific band. (B) Addition of the anti-MTA1 or Snail antibodies resulted in a supershifted band (indicated by an arrow). Addition of the anti-Slug antibody decreased the formation of the supershifted band.

Article Snippet: The MTA1 siRNA (SC35981), HDAC1 siRNA (SC44208), and Slug siRNA (SC38393) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Palmatine Attenuates Metastatic Lung Colonization of Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2022.853230

Figure Lengend Snippet: Effect of palmatine on Lung metastasis. Data represents mean ± SEM. n = 10, One-way analysis of variance followed by Dunnett’s post hoc test. Lung arterial blood gas: (A) potential of hydrogen (pH), (B) partial oxygen pressure (PaO 2 ), (C) partial carbon dioxide pressure (PaCO 2 ), (D) bicarbonate (HCO 3 ). (A) ns (not significant) vs. Saline treated and Lung metastasis groups (B) ++++ p < 0.0001 vs. Saline treated group and ns (not significant), ** p = 0.002, **** p < 0.0001 vs. Lung metastasis group (C) ++++ p < 0.0001 vs. Saline treated group and * p = 0.025, **** p < 0.0001 vs. Lung metastasis group (D) ++++ p < 0.0001 vs. Saline treated group and * p = 0.0127, **** p < 0.0001 vs. Lung metastasis group. Lung acid-base values: (E) base excess (BE) (F) base excess extracellular fluid (BE ecf) (G) anion gap (H) sodium (Na + ) (I) potassium (K + ) (J) Chloride (Cl − ). (E–G) ++++ p < 0.0001 vs. Saline treated group and ns (not significant), **** p < 0.0001 vs. Lung metastasis group (H) +++ p = 0.0002 vs. Saline treated group and ns (not significant), * p = 0.0112, **** p < 0.0002 vs. Lung metastasis group (I) ++++ p < 0.0001 vs. Saline treated group and ** p = 0.0016, **** p < 0.0001 vs. Lung metastasis group (J) ns (not significant) vs. Saline treated and Lung metastasis group. Lung immunohistochemistry: (K) lung histology scores (L) lung MTA1 expression (M) lung p53 expression. (K) ++++ p < 0.0001 vs. Saline treated groups and ns (not significant), *** p = 0.001, **** p < 0.0001 vs. Lung metastasis group. (L) ++++ p < 0.0001 vs. Saline treated groups and **** p < 0.0001 vs. Lung metastasis group. (M) ns (not significant) vs. Saline treated group and ns (not significant), ** p = 0.002, *** p = 0.0007, **** p < 0.0001 vs. Lung metastasis group.

Article Snippet: Roswell Park Memorial Institute 1640 medium, Trypsin/EDTA, l -glutamine (Pan Biotech, Aidenbach, Germany), Fetal bovine serum, Penicillin-streptomycin (Capricorn scientific, Ebsdorfergrund, Germany), MTA1 (Cusabio, Houston, USA) and p53 Antibodies (Sigma Aldrich, St. Louis, USA).

Techniques: Saline, Immunohistochemistry, Expressing

Journal: Frontiers in Pharmacology

Article Title: Palmatine Attenuates Metastatic Lung Colonization of Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2022.853230

Figure Lengend Snippet: Representative photomicrographs of MTA1 expression in IHC sections of the lung tissue: (A) Normal saline (B) Lung metastasis (C–E) Palmatine 1–10 mg/kg (F) Doxorubicin 5 mg/kg. Data were obtained from five fields per slide with seven slides analyzed from each group. Red arrows show immunoreactive areas. Scale bar 20 mm (×10, (A–F) .

Article Snippet: Roswell Park Memorial Institute 1640 medium, Trypsin/EDTA, l -glutamine (Pan Biotech, Aidenbach, Germany), Fetal bovine serum, Penicillin-streptomycin (Capricorn scientific, Ebsdorfergrund, Germany), MTA1 (Cusabio, Houston, USA) and p53 Antibodies (Sigma Aldrich, St. Louis, USA).

Techniques: Expressing, Saline

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Transcriptional levels of MTA1 in breast cancer tissues and adjacent nontumoral breast tissues from the GEO database (GSE1477). The box plots show the medians (black lines), 25th and 75th percentiles (boundaries), and minimum/maximum values (whiskers). The p value is indicated. (b) MTA1 positively correlates with overall survival in human breast cancer tissues. Datasets used for overall survival analysis are from indicated GEO datasets. Red lines represent the survival probability of patients with cancer with high MTA1 expression levels and black lines represent that with low MTA1 expression levels. HR, hazard ratio. The p values are indicated. (c) PCC R value revealed a moderate positive correlation between MTA1 and OGT in TCGA and GTEx datasets samples.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Breast cancer cells with intrinsic and acquired Adm adaptation contain higher levels of MTA1 and OGT compared with Adm sensitive cells. The expression of indicated proteins in breast cancer cells were analysed by immunoblotting. (b) The PPIs between MTA1, OGT and NuRD components are increased in MDA-MB-231 and MCF-7/ADR cells compared with MCF-7 cells. MTA1 immunoprecipitation was performed and the immunoprecipitated fractions were analysed by immunoblotting by indicated antibodies. (c) The inhibition of O-GlcNAc modification increases the sensitivity of breast cancer cells to Adm and restrains the interaction of MTA1 with OGT and NuRD complex. MCF-7/ADR cells were transfected with OGT siRNA (siOGT) or treated with 100 μM L01 and then incubated with indicated doses of Adm for 48 h. The protein levels were examined by immunoblotting. Vehicle, cells treated with DMSO. The cell viability was assessed through an CCK8 assay. (d) The activation of O-GlcNAc modification increases the adaptation of MCF-7 cells to Adm and elevates the interaction of MTA1 with OGT and NuRD complex. MCF-7 cells were transfected with OGT expression plasmid (pcDNA-OGT) or treated with 100 μM PugNAc and then incubated with indicated doses of Adm for 48 h. The protein levels were examined by immunoblotting. Vehicle, cells treated with DMSO. The cell viability was assessed through an CCK8 assay.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Modification, Transfection, Incubation, CCK-8 Assay, Activation Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Schematic of the metabolic labeling (GalNAz) based O-GlcNAc sites identification and quantification proteomics method. MCF-7 and MCF-7/ADR cells were incubated on 1mM GalNAz media for 24 h. Chromatin was crosslinked and conjugated to a cleavable DADPS alkyne-biotin linker by a downstream click reaction. Biotin labeled chromatin proteins were enriched with streptavidin beads and subsequently digested with Trypsin. After cleavage of DADPS by formic acid, O-GlcNAc modified peptides were quantitative analysis using quantitative LC-MS/MS. (b) Volcano plot of quantitative proteomics data of O-GlcNAc chromatin-associated proteins in MCF-7 and MCF-7/ADR cells (n = 5 biologically independent experiments). The component of NuRD complex mentioned in this study are labeled. (c) Schematic representation of full-length MTA1 and its potential O-GlcNAc sites (predicted by YinOYang 1.2 server). (d) MTA1 is O-GlcNAcylated at S237/S241/S246. MCF-7/ADR cells expressing HA-WT-MTA1, HA-MTA1-S237A, HA-MTA1-S241A, HA-MTA1-S246A, or HA-MTA1-S237A/S241A/S246A (HA-MTA1-3A) were immunoprecipitated with anti-HA magnetic beads. O-GlcNAc modification was analyzed by immunoblotting. Vehicle, cells transfected with empty vector. (e) The O-GlcNAc levels of MTA1 is decreased after O-GlcNAc sites mutation. Immunoprecipitation was performed using O-GlcNAc-binding lectin sWGA, and the immunoprecipitated fractions were analyzed by immunoblotting for MTA1. Vehicle, cells transfected with empty vector. (f) After treatment with 100 μM L01 or PugNAc for 24 h, MCF-7/ADR cells expressing HA-MTA1-WT or HA-MTA1-3A were immunoprecipitated with anti-HA magnetic beads. Indicated proteins were analyzed by immunoblotting.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Labeling, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Coverage plot of the MTA1 ChIP-seq peak locations over the whole human genome. The peaks of HA-MTA1-WT and HA-MTA1-3A stably expressed MCF-7 cells showed distinct characteristic. (b) HA-MTA1-WT and HA-MTA1-3A ChIP-seq peak annotation relative to known genomic elements. (c) Heat maps of the ChIP-seq signal density at TSSs (±3 kb). The average signal profile is shown. The red color indicates low a signal, and a blue color indicates a high signal. (d) Average enrichment profiles of published H3K27ac, H3K4me3 (GSE97481), H3K27me3 (GSE96363) and H3K4me1 (GSE86714) ChIP-seq reads at HA-MTA1-WT and HA-MTA1-3A ChIP-seq peaks.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, Stable Transfection

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Normalization and quantitative comparisons of ChIP-seq peaks using MAnorm. MA plots of all peaks from both HA-MTA1-WT and HA-MTA1-3A ChIP-seq datasets after MAnorm. Differential quantitative peaks (“MTA1-WT-biased” or “MTA1-3A-biased”, M value (log2 fold change) ≥ 1 or ≤ -1 and P value ≤ 10 −5 ) are shown. (b) The differential quantitative peak associated genes were used for GO term analysis. (c) Scatter plot showing the RNA-seq DEGs expression levels (fragments per kilobase of transcript per million mapped reads (FPKM), fold change ≥ 1.5, FDR ≤ 0.05) in HA-MTA1-WT expressed MCF-7 cells compared with HA-MTA1-3A expressed MCF-7 cells. n = 2 biologically independent RNA-seq replicates. (d) Left images: GO analysis of RNA-seq DEGs in HA-MTA1-WT and HA-MTA1-3A expressed MCF-7 cells. The gene numbers identified in certain terms are shown. Right images: GSEA of RNA-seq DEGs. The normalized enrichment score (NES) and p value are indicated. (e) Overlap of 2073 differential quantitative peak associated genes identified from ChIP-seq with 950 DEGs in RNA-seq is shown in a Venn diagram. The 95 overlapping genes were used for GO term analysis. (f) The rescue expression of HA-MTA1-WT and HA-MTA1-3A were performed in MCF-7/ADR-shMTA1 cells (shMTA1+MTA1-WT and shMTA1+MTA1-3A, respectively). Cells were subsequently treated with increasing doses of Adm for 48 h. Vehicle, cells transfected with empty vector. The cell viability was assessed using a CCK8 assay. The protein levels of MTA1 were analyzed by immunoblotting.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, RNA Sequencing, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Western Blot

Journal: bioRxiv

Article Title: O-GlcNAc regulates MTA1 transcriptional activity during breast cancer cells genotoxic adaptation

doi: 10.1101/2021.02.08.430201

Figure Lengend Snippet: (a) Left images: Integrative Genomics Viewer (IGV) tracks showing ChIP-seq signal at the promoter regions of c-Fos . Right images: Validation of O-GlcNAc MTA1 binding peaks by ChIP-qPCR. Each bar represents the fold enrichment of binding relative to the input. IgG and random primers that could not specifically bind the indicated gene promoter regions (off target) were used as negative controls. Mock, MCF-7 cells transfected with empty vector. (b) MCF-7/ADR cells were transfected with a reporter vector consisting of luciferase cDNA fused to the c-Fos promoter. The pGL3-basic vector (Vehicle) was used as a control. (c) Effect of MTA1 O-GlcNAc modification on c-Fos transcription and protein levels in MCF-7/ADR cells. The gene mRNA levels were analyzed by qPCR. The protein levels were analyzed by immunoblotting. Vehicle, cells transfected with empty vector. (d) MCF-7/ADR cells were transfected with c-Fos siRNA and then incubated with indicated doses of Adm for 48 h. Vehicle, cells treated with DMSO. The cell viability was assessed through an CCK8 assay. The protein levels were examined by immunoblotting. (e) Proposed model for the MTA1 transcriptional activity regulated by O-GlcNAc modification during breast cancer cells genotoxic adaptation. O-GlcNAc modification enhances the interaction of MTA1 with NuRD complex and chromatin. O-GlcNAc MTA1 preferentially modulates target genes transcription and drives breast cancer cells adaptation during genotoxic stimulation.

Article Snippet: OGT siRNA (#sc-40781), c-Fos siRNA (#sc-29221) and MTA1 shRNA (#sc-35981-SH) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, Biomarker Discovery, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation, Luciferase, Control, Modification, Western Blot, Incubation, CCK-8 Assay, Activity Assay

Journal: American Journal of Cancer Research

Article Title: Nuclear moonlighting of the secreted growth factor heregulin drives endocrine-resistant breast cancer independently of HER2/HER3 signaling

doi:

Figure Lengend Snippet: The isolated deficiency of nuclear heregulin-β2 but not of secreted heregulin-β2 to bind HER receptors abolishes heregulin-β2-driven endocrine resistance in vitro and in vivo. A. Estradiol (E2)-depleted cells were plated in soft agarose containing E2 (10-9 mol/L), tamoxifen (10-7 mol/L), fulvestrant (10-7 mol/L), their combinations, or ethanol (v/v) or DMSO (v/v) vehicle only for 7-10 days. Colony formation (≥ 60 μm) was assessed using a colony counter. Each experimental value represents the mean colony number (columns) ± S.D. (bars) from at least three separate experiments in which triplicate dishes were counted. B. Representative immunoblotting analyses showing the expression of P-HER/total HER, P-AKT/total AKT, P-MAPK/total MAPK, CYR61, ERα, and MTA1, in cells cultured in the absence and presence of serum. C. Shown are the mean tumor volumes (±SD) in MCF-7/pBABE, MCF-7/HRG, MCF-7/HRG∆NLS, MCF-7/HRG∆EGF, and MCF-7/HRG∆EGF + ∆NLS xenograft-bearing nude mice following treatment with estradiol, tamoxifen (TAM), fulvestrant (FVT), or their combinations for seven weeks (n = 10 animals/experimental group).

Article Snippet: Goat polyclonal anti-MTA1 antibody (45-094) was purchased from ProSci (Poway, CA).

Techniques: Isolation, In Vitro, In Vivo, Western Blot, Expressing, Cell Culture

Journal: American Journal of Cancer Research

Article Title: Nuclear moonlighting of the secreted growth factor heregulin drives endocrine-resistant breast cancer independently of HER2/HER3 signaling

doi:

Figure Lengend Snippet: Nuclear moonlighting of heregulin-β2 drives endocrine resistance in breast cancer: A working model. A. Isolated deficiency of nuclear HRG abolishes the HRG-driven endocrine resistance phenotype in vitro and in mouse xenograft models, while preserving its capacity to activate the HRG/HER/MAPK autocrine signaling axis. Conversely, isolated deficiency of secreted HRG to bind HER receptors does not impair endocrine resistance. B. Left. Nucleolin is a/the key driver of HRG nuclear shuttling in breast cancer cells. HRG interacts with nucleolin via a nuclear localization signal motif located at the N-terminal extracellular domain of HRG. Nucleolin interacts with HRG via aspartate/glutamate-rich acidic stretches located at the N-terminal domain of nucleolin. Right. MTA proteins (MTA1/2) are part of the chromatin and histone deacetylation complex (NuRD), which contains histone deacetylases (HDAC1/2) and functions in chromatin remodeling and histone deacetylation to drive biologically aggressive cancer phenotypes. MTA1 can directly interact with the ligand-binding domain of the ERα and nuclear HRG can stimulate the association of MTA1 with HDAC2 on the chromatin site of an ER-responsive element (ERE) in the promoter regions of estrogen responsive genes [32]. The repressive function of MTA1 protein on ERα is mediated via histone deacetylation by HDAC1/2, thereby suggesting that nuclear HRG might enhance/stabilize the potent co-repressor epigenetic function of NuRD during the trans-activation of ERα. The MTA1-containing NuRD complex can deacetylate also non-histone proteins such as the tumor-suppressor PTEN. In the presence of nuclear HRG, the MTA1/HDAC complex might be stabilized and localized in the nucleus where it can bind to acetylated PTEN. The MTA1/HDAC complex drives deacetylation (inactivation) of PTEN, which can diffuse into the cytoplasmic compartment but lacking a tumor suppressive function against phospho-active AKT. The isolated deficiency of nuclear HRG suffices to down-regulate MTA1 and disrupt MTA1/HDAC functionality, thereby increasing the acetylated (active) form of PTEN that can then inhibit AKT activity while preserving the HRG capacity to activate the HER/MAPK autocrine signaling axis. The ability of nuclear HRG to drive alterations in acetylation of both histone and non-histone proteins via regulation of the master co-regulatory molecule MTA-1 might explain how the nucleolin-driven nuclear moonlighting of HRG uncouples its well-known role as a driver of endocrine resistance from its canonical HER receptor-activating role in breast cancer cells.

Article Snippet: Goat polyclonal anti-MTA1 antibody (45-094) was purchased from ProSci (Poway, CA).

Techniques: Isolation, In Vitro, Preserving, Ligand Binding Assay, Activation Assay, Activity Assay