Journal: Antioxidants

Article Title: ABCA1 Transporter Is Involved in the Secretion of CuZn Superoxide Dismutase (SOD)-1 by Activated Human T Lymphocytes

doi: 10.3390/antiox14121487

Figure Lengend Snippet: ABC-transporter-blocking by glyburide treatment significantly affects SOD-1 content of culture supernatants from PBMC ON cultured in the presence of medium or anti-CD3 moAb to mimic antigen-dependent T cell activation. Western blotting was performed on intracellular and extracellular protein fractions obtained from PBMC cultured ON in medium alone or stimulated with anti-CD3 monoclonal antibody, in the absence or presence of glyburide (20 μM and 30 μM). ( Left ) Densitometric quantification of intracellular SOD-1 normalised to total ERK. A significant increase in intracellular SOD-1 content was observed following T cell activation; glyburide treatment was associated with an increasing trend of intracellular SOD-1 content. ( Right ) Analysis of extracellular SOD-1 from culture supernatants, normalised to total cellular protein content. Activated lymphocytes showed a significant increase in extracellular SOD-1, as compared with the unstimulated cells; as shown, glyburide treatment at both 20 μM and 30 μM concentration significantly reduced the SOD-1 amount. Data are expressed as mean ± SEM in six independent experiments. White, grey, grey-dotted, and grey-striped columns indicate data obtained from supernatants of PBMC cultured with medium, activated with anti-CD3 alone or in the presence of glyburide at 20 μM and 30 μM, as indicated. Statistical analysis was performed by using one-way ANOVA; statistical significance values are indicated.

Article Snippet: To investigate the mechanisms involved in both basal and activation-dependent SOD-1 secretion by T lymphocytes, overnight (ON) cultures were set up in RPMI 1640 medium supplemented with 0.2% FBS (Thermo Fisher Scientific, Waltham, MA, USA), alone, in the presence of an activating concentration of the anti-CD3 mAb, as well as with 20 or 30 μM of the ABC transporter inhibitor glyburide (glibenclamide), purchased from Selleck Chemicals, Houston, TX, USA.

Techniques: Blocking Assay, Cell Culture, Activation Assay, Western Blot, Concentration Assay

Journal: Antioxidants

Article Title: ABCA1 Transporter Is Involved in the Secretion of CuZn Superoxide Dismutase (SOD)-1 by Activated Human T Lymphocytes

doi: 10.3390/antiox14121487

Figure Lengend Snippet: The quantitative analysis of extracellular SOD-1 content by ELISA detection shows a significant decrease in the enzyme in supernatants from PBMC ON cultured in the presence of anti-CD3 moAb to mimic antigen-dependent T cell activation. PBMC were cultured ON in the presence of medium, or activated with anti-CD3 monoclonal antibody, in the absence or presence of glyburide (20 μM, 30 μM), as indicated. Culture supernatants were analysed for SOD-1. Data are expressed as mean ± SEM obtained in five independent experiments. White, grey, grey-dotted, and grey-striped columns indicate data obtained from PBMC ON cultured in medium, activated with anti-CD3 alone or in the presence of glyburide at 20 μM and 30 μM, respectively. Statistical analysis has been performed by using Mann–Whitney test; statistical significance values are indicated.

Article Snippet: To investigate the mechanisms involved in both basal and activation-dependent SOD-1 secretion by T lymphocytes, overnight (ON) cultures were set up in RPMI 1640 medium supplemented with 0.2% FBS (Thermo Fisher Scientific, Waltham, MA, USA), alone, in the presence of an activating concentration of the anti-CD3 mAb, as well as with 20 or 30 μM of the ABC transporter inhibitor glyburide (glibenclamide), purchased from Selleck Chemicals, Houston, TX, USA.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, MANN-WHITNEY

Journal: Antioxidants

Article Title: ABCA1 Transporter Is Involved in the Secretion of CuZn Superoxide Dismutase (SOD)-1 by Activated Human T Lymphocytes

doi: 10.3390/antiox14121487

Figure Lengend Snippet: Flow cytometry analysis of intracellular SOD-1 in T and “non-T” lymphocyte populations following antigen-dependent T cell activation by anti-CD3 triggering, alone or in the presence of glyburide treatment, shows a significant increase in SOD-1 intracellular content only in the T cell subset. PBMC were cultured ON in medium alone or stimulated with anti-CD3 monoclonal antibody, in the absence or presence of glyburide (20 μM and 30 μM). Intracellular SOD-1 levels were detected by immunofluorescence and flow cytometry. ( A ) Gating strategy to identify the T cell and the “non-T” cell subsets. ( B ) Cumulative data from four independent experiments; SOD-1 content has been shown as the ratio of mean intensity fluorescence (MIF) value for the T and the “non-T” cell subset and the control MIF value obtained after staining the same cell population with the isotype control mAb, as described . Statistical evaluation of data has been performed by means of the Wilcoxon matched-pairs rank test. Statistical significance values are indicated ( C , D ). Representative binding profiles showing SOD-1 intracellular amount in T cells after anti-CD3 activation and its further enhancement following glyburide treatment. Data are expressed as mean ± SEM. Light blue, dotted light blue, striped light blue lines represent T cells and “non-T” subset; red, dotted yellow and striped green represent SOD-1 binding in T cells activated in the absence or in the presence of glyburide at 20 μM or 30 μM concentration, respectively. Statistical analysis has been performed by using Wilcoxon matched-pairs rank test; statistical significance values are indicated.

Article Snippet: To investigate the mechanisms involved in both basal and activation-dependent SOD-1 secretion by T lymphocytes, overnight (ON) cultures were set up in RPMI 1640 medium supplemented with 0.2% FBS (Thermo Fisher Scientific, Waltham, MA, USA), alone, in the presence of an activating concentration of the anti-CD3 mAb, as well as with 20 or 30 μM of the ABC transporter inhibitor glyburide (glibenclamide), purchased from Selleck Chemicals, Houston, TX, USA.

Techniques: Flow Cytometry, Activation Assay, Cell Culture, Immunofluorescence, Fluorescence, Control, Staining, Binding Assay, Concentration Assay

Journal: Antioxidants

Article Title: ABCA1 Transporter Is Involved in the Secretion of CuZn Superoxide Dismutase (SOD)-1 by Activated Human T Lymphocytes

doi: 10.3390/antiox14121487

Figure Lengend Snippet: SOD1 and ABCA1 co-localise in human activated T lymphocytes. ( A ). Representative confocal images displaying SOD-1 (red) and ABCA1 (green) immunoreactivities in human T lymphocytes ON cultured under control condition (medium, ( a – c )) and after activation with anti-CD3 moAb in the absence ( d – i ) or in presence of 30 μM glyburide ( j – o ). Panels ( g – i ) show high-magnification views of the boxed areas in ( d – f ), respectively. Arrowheads in ( g – i ) point to cells with a clear SOD-1 immunosignal along the plasma membrane. Arrowheads in ( m – o ) point to cells with SOD-1 cytoplasmic immunoreactivity. Scale bars: ( a – f ) and ( j – l ), 10 μm; ( g – i ) and ( m – o ), 5 μm. ( B ). Quantitative analysis of SOD-1-immunoreactive area in T lymphocyte cultures under control conditions and after activation, in the absence or in the presence of 30 μM glyburide ON exposure. The values represent the mean ± SEM; (n = 3) * p < 0.05 vs. medium. ( C ). Quantitative analysis of ABCA1 fluorescence intensity in T lymphocyte cultures under control conditions, and after activation, in the absence or in the presence of 30 μM glyburide. The values represent the mean ± SEM (n = 3). * p < 0.05 vs. medium; ˄ p < 0.05 vs. anti-CD3. ( D ). Line profiling of SOD-1 (red) and ABCA1 (green) fluorescence intensities along the line selected on the plasma membrane of anti-CD3 activated T cells.

Article Snippet: To investigate the mechanisms involved in both basal and activation-dependent SOD-1 secretion by T lymphocytes, overnight (ON) cultures were set up in RPMI 1640 medium supplemented with 0.2% FBS (Thermo Fisher Scientific, Waltham, MA, USA), alone, in the presence of an activating concentration of the anti-CD3 mAb, as well as with 20 or 30 μM of the ABC transporter inhibitor glyburide (glibenclamide), purchased from Selleck Chemicals, Houston, TX, USA.

Techniques: Cell Culture, Control, Activation Assay, Clinical Proteomics, Membrane, Fluorescence

Journal: Antioxidants

Article Title: ABCA1 Transporter Is Involved in the Secretion of CuZn Superoxide Dismutase (SOD)-1 by Activated Human T Lymphocytes

doi: 10.3390/antiox14121487

Figure Lengend Snippet: Distributionanalysis of SOD-1 and ABCA3 in human activated T lymphocytes . ( A ). Representative confocal images displaying SOD-1 (red) and ABCA3 (green) immunoreactivities in human T lymphocytes under control condition (medium, ( a – c )) and after ON activation with anti-CD3 in the absence ( d – i ) or in the presence of 30 μM glyburide ( j – o ). Panels ( g – i ) show high-magnification views of the boxed areas in ( d – f ), respectively. Arrowheads in ( g – i ) point to cells with a clear SOD-1 immunosignal along the plasma membrane. Arrows in ( m – o ) point to cells with SOD-1 cytoplasmic immunoreactivity. Scale bars: ( a – f ) and ( j – l ): 10 μm; ( g – i ) and ( m – o ), 5 μm. ( B ). Quantitative analysis of SOD-1-immunoreactive area in T lymphocytes cultured under control conditions and after activation with anti-CD3 moAb in the absence or in the presence of 30 μM glyburide. The values represent the mean ± SEM (n = 3). * p < 0.05 vs. control. ( C ). Quantitative analysis of ABCA3 fluorescence intensity in T lymphocyte cultures under control conditions and after ON activation with anti-CD3, in the absence or in the presence of 30 μM glyburide. The values represent the mean ± SEM. (n = 3). ( D ). Line profiling of SOD-1 (red) and ABCA3 (green) fluorescence intensities along the line selected on the cell plasma membrane of T cells activated with anti-CD3 ( i ) in the absence of 30 μM glyburide.

Article Snippet: To investigate the mechanisms involved in both basal and activation-dependent SOD-1 secretion by T lymphocytes, overnight (ON) cultures were set up in RPMI 1640 medium supplemented with 0.2% FBS (Thermo Fisher Scientific, Waltham, MA, USA), alone, in the presence of an activating concentration of the anti-CD3 mAb, as well as with 20 or 30 μM of the ABC transporter inhibitor glyburide (glibenclamide), purchased from Selleck Chemicals, Houston, TX, USA.

Techniques: Control, Activation Assay, Clinical Proteomics, Membrane, Cell Culture, Fluorescence