Journal: International Journal of Molecular Sciences

Article Title: Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes

doi: 10.3390/ijms23031169

Figure Lengend Snippet: ALA treatment enhances in vitro Treg differentiation. Naïve CD4 T cells were isolated from female NOD mice and then cultured in anti-CD3 antibody coated (1 μg/mL) plate and combined with anti-CD28 antibody (1 μg/mL) plus human IL-2 cytokine (5 ng/mL) and TGF-β (5 ng/mL) with either PBS or different concentrations of ALA (10 μM, 50 μΜ, or 100 μM). ( A ) Representative plots of flow cytometry of in vitro Treg differentiation with or without ALA treatment. ( B ) The percentage of Tregs was significantly increased in the stimulation medium that contained ALA (n = 3; 0 μM vs. 10 μM p = 0.0004, vs. 50 μM p = 0.0002, vs. 100 μM p = 0.0017). ( C ) Representative figure of Western blot test of STAT5 and phosphorylated-STAT5 (p-STAT5). Alpha-tubulin was used as internal control. ( D ) The ratio between p-STAT5 and total STAT5 after normalization with α-tubulin. The ratio between p-STAT5 and total STAT5 was significantly increased in the stimulation medium that contained ALA (n = 3; 0 μM vs. 10 μM p = 0.0189, vs. 50 μM p = 0.0032, vs. 100 μM p = 0.0129). Data are expressed as means ± SEM. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The membrane was blocked with 5% skim milk at room temperature for 1 h and was then incubated in a buffer with a rabbit anti-STAT5 antibody (GeneTex Inc., Alton Pkwy Irvine, CA, USA), anti-acetylated H3 (GeneTex Inc.) or a mouse anti-β-actin antibody (GeneTex Inc.) overnight.

Techniques: In Vitro, Isolation, Cell Culture, Flow Cytometry, Western Blot, Control

Journal: Cells

Article Title: Glucocorticoid-Induced Leucine Zipper-Mediated TLR2 Downregulation Accounts for Reduced Neutrophil Activity Following Acute DEX Treatment

doi: 10.3390/cells10092228

Figure Lengend Snippet: GILZ and STAT5 staining overlaps in mouse neutrophils. ( A ) Isolated peripheral WT and KO neutrophils were spotted on slides after 3 h of in vivo DEX treatment. The nuclei were stained with DAPI. Cytoplasmic and nuclear STAT5 (green) and cytoplasmic GILZ (red) staining showed overlapping spots (yellow, Merge 1) in DEX-treated cells (middle panels). Overlapping staining of STAT5, GILZ, and DAPI is shown in Merge 2. GILZ staining was absent in KO neutrophils (lower panels). ( B ) Cells with both cytoplasmic and nuclear (n + c) STAT5 staining were counted and expressed as a percentage of total cell number. Values represent mean ± SD. Scale bar, 10 µm. All experiments were repeated three times. The results of a representative experiment are presented. * p < 0.05.

Article Snippet: The slides were then incubated with a rabbit anti-STAT5 antibody (Elabscience, ESAP12240) or rabbit NF-κB (Cell Signaling) and goat anti-GILZ Ab (S-15; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C.

Techniques: Staining, Isolation, In Vivo

Journal: Cells

Article Title: Glucocorticoid-Induced Leucine Zipper-Mediated TLR2 Downregulation Accounts for Reduced Neutrophil Activity Following Acute DEX Treatment

doi: 10.3390/cells10092228

Figure Lengend Snippet: GILZ and STAT5 interact in DEX-treated neutrophils. ( A ) An in-situ proximity ligation assay in isolated peripheral WT and KO neutrophils of in vivo DEX-treated mice. Red spots indicate a GILZ/STAT5 interaction: low number of spots were observed in the WT untreated mice (two upper rows of panels), whereas a higher number of spots were identified in DEX-treated cells (two lower rows of panels). ( B ) Statistical analysis of the spot number was performed using ICY free software. Two fields of representative stainings are shown (WT and WT+DEX). Values represent the means ± SD of more than 20 fields. Scale bar: 10 µm. ** p < 0.01.

Article Snippet: The slides were then incubated with a rabbit anti-STAT5 antibody (Elabscience, ESAP12240) or rabbit NF-κB (Cell Signaling) and goat anti-GILZ Ab (S-15; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C.

Techniques: In Situ, Proximity Ligation Assay, Isolation, In Vivo, Software