Journal: Journal of Immunology Research

Article Title: HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response

doi: 10.1155/2018/2942679

Figure Lengend Snippet: Protein expression of MTA1 by Western Blot analysis. The protein expression of MTA1 (81 kDa) was evaluated in four human cancer cell lines. β -Actin (42 kDa) was used as loading control, and rabbit polyclonal anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China) was used in this assay. (a) The bands representing MTA1 and β -actin in each cancer cell line. (b) Gray analysis of the MTA1 protein band intensities (relative to the β -actin band intensity) was carried out by Image J.

Article Snippet: Further, the membranes were incubated overnight at 4°C with the indicated primary antibody—rabbit anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China/cat: 5646, dilutions: 1 : 500, Cell Signaling Technology, USA) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China)—and then washed three times in TBST.

Techniques: Expressing, Western Blot

Journal: Journal of Immunology Research

Article Title: HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response

doi: 10.1155/2018/2942679

Figure Lengend Snippet: IFN- γ release and lyse target cell ability of peptide-induced CTLs. PBMCs from six HLA-A ∗ 02 + healthy donors were collected and induced by the indicated peptides (10 μ g/ml) three times in RPMI 1640 medium supplemented with 3 μ g/ml β 2-M (once at each stimulation), interleukin-2 (50 U/ml, on day 3 and 1 day after each stimulation), and 10% FCS for 21 days. CTLs induced by COX-2 321–329 were used as positive controls. On day 21, the CTLs were collected, and the IFN- γ secretion and cytotoxic activity of the CTLs were measured. (a) CTL secretion of IFN- γ was detected by the ELISPOT assay ( n = 6). T2 cells loaded with/without peptide (50 μ g/ml) for 4 h were used as the stimulating cells. LDH cytotoxicity assays were performed ( n = 3) using the following target cell lines: (b) SW620 (HLA-A2 + , MTA1 + ), (c) MDA-MB-231 (HLA-A2 + , MTA1 + ), (d) SW620 incubated with an anti-human HLA-A2 antibody (HLA-A2 − , MTA1 + ), (e) HT-29 (HLA-A2 − , MTA1 + ), (f) MCF-7 (HLA-A2 + , MTA1 + ), (g) HUVEC (HLA-A2 + , MTA1 low ), and (h) Het-1A (HLA-A2 + , MTA1 low ). CTLs induced by PBS were used as the negative control. Data represent means ± standard deviation (SD), ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the PBS group.

Article Snippet: Further, the membranes were incubated overnight at 4°C with the indicated primary antibody—rabbit anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China/cat: 5646, dilutions: 1 : 500, Cell Signaling Technology, USA) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China)—and then washed three times in TBST.

Techniques: Activity Assay, Enzyme-linked Immunospot, Incubation, Negative Control, Standard Deviation

Journal: Journal of Immunology Research

Article Title: HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response

doi: 10.1155/2018/2942679

Figure Lengend Snippet: Specific lysis of target cells and secretion of IFN- γ by induced CTLs in immunizing HLA-A2.1/K b transgenic mice immunized with the indicated peptides. Peptide (100 μ g) and the IA b -restricted HBV-core 128 T helper epitope (140 μ g) emulsified in IFA were used to immunize HLA-A2.1/K b transgenic mice on days 0, 5, and 10. PBS only or T helper peptide emulsified in IFA was used as the negative control. On day 11, we isolated splenocytes from the mice and restimulated the cells with 10 μ g/ml peptide and 10 U/ml rmIL-2 to repriming these splenocytes in vitro for additional 6 days. An LDH assay was performed to assess the lysis activity of the induced CTLs toward (a) SW620 cells (HLA-A2 + , MTA1 + ), (b) T2 cells pulsed with each peptide, and (c) SW620 cells incubated with an anti-human HLA-A2 antibody (HLA-A2 − , MTA1 + ). IFN- γ secretion was detected by the ELISPOT assay. (d) T2 cells incubated with each peptide and an irrelevant peptide (50 μ g/ml) for 4 h were used as the stimulating cells. Each sample was measured in triplicate. Data represent means ± SD ( n = 5). ∗ P < 0.05, ∗∗ P < 0.05, and ∗∗∗ P < 0.001 vs. the T helper epitope group.

Article Snippet: Further, the membranes were incubated overnight at 4°C with the indicated primary antibody—rabbit anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China/cat: 5646, dilutions: 1 : 500, Cell Signaling Technology, USA) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China)—and then washed three times in TBST.

Techniques: Lysis, Transgenic Assay, Negative Control, Isolation, In Vitro, Lactate Dehydrogenase Assay, Activity Assay, Incubation, Enzyme-linked Immunospot

Journal: Journal of Immunology Research

Article Title: HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response

doi: 10.1155/2018/2942679

Figure Lengend Snippet: Specific T cells induced by MTA1 (1–283) secrete IFN- γ and lyse target cells. PBMCs from healthy HLA-A ∗ 02 + donors were isolated and stimulated with protein MTA1 (1–283) (at final concentrations of 0, 1, 5, 10, 30, and 50 μ g/ml) in RPMI 1640 medium supplemented with 50 U/ml interleukin-2 and 10% FCS once a week for 21 days. On day 21, the induced T cells were collected; (a) IFN- γ secretion was assessed by the ELISPOT assay; ∗ P < 0.05 and ∗∗∗ P < 0.001 between two groups. (b) Cytotoxic activity was assessed by the LDH assay using SW620 cells (HLA-A2 + , MTA1 + ) as the target cells. T cells induced by PBS were used as negative controls. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control group. Data represent means ± SD ( n = 4).

Article Snippet: Further, the membranes were incubated overnight at 4°C with the indicated primary antibody—rabbit anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China/cat: 5646, dilutions: 1 : 500, Cell Signaling Technology, USA) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China)—and then washed three times in TBST.

Techniques: Isolation, Enzyme-linked Immunospot, Activity Assay, Lactate Dehydrogenase Assay

Journal: Journal of Immunology Research

Article Title: HLA-A2-Restricted Epitopes Identified from MTA1 Could Elicit Antigen-Specific Cytotoxic T Lymphocyte Response

doi: 10.1155/2018/2942679

Figure Lengend Snippet: Specific lysis of various cell lines and IFN- γ secretion by induced T cells in HLA-A2.1/K b transgenic mice immunized with MTA1 (1–283) . MTA1 (1–283) (100 μ g/ml) or the same volume of PBS (negative control) emulsified in IFA was used to immunize HLA-A2.1/K b transgenic mice on days 0 and 7. On day 14, the mice were sacrificed, and their splenocytes were collected and restimulated for another 6 days with the peptide pool (10 μ g/ml) and 50 U/ml rmIL-2 in vitro . (a) Serum was collected from the immunized mice on day 14, and the serum IFN- γ concentration was measured by ELISA. (b) The percentage of IFN- γ secreting peptide-specific CD8 + T cells induced by MTA1 (1–283) and PBS was analyzed by an intracellular staining assay. (c) The percentages of IFN-γ + CD4 + T cells in the MTA1 (1–283) and PBS groups were determined. (d) Specific lysis of target cells SW620 cells (HLA-A2 + , MTA1 + ) was assessed by the LDH cytotoxicity assay. ∗ P < 0.05 and ∗∗∗ P < 0.001. P < 0.05 vs. the control group. Data represent means ± SD ( n = 5).

Article Snippet: Further, the membranes were incubated overnight at 4°C with the indicated primary antibody—rabbit anti-MTA1 antibody (cat: bs-1412R, dilutions: 1 : 1500, Bioss Antibodies, China/cat: 5646, dilutions: 1 : 500, Cell Signaling Technology, USA) or rabbit polyclonal anti- β -actin antibody (cat: bs-0061R, dilutions: 1 : 50,000, Bioss Antibodies, China)—and then washed three times in TBST.

Techniques: Lysis, Transgenic Assay, Negative Control, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, LDH Cytotoxicity Assay

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: 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