4 p stat3

Naïve CD4⁺ T cells were isolated from spleen and lymph nodes of C57BL/6 mice using naïve CD4 negative selection kit (StemCell) and activated under Th17-polarizing conditions in the presence of irradiated wild-type splenocytes at a 5:1 ratio, with anti-CD3 (2.5 μg/ml) (145-2C11, BioXCell), anti-IL-4 (10 μg/ml) (11B11, BioXCell), anti-IFNγ (10 μg/ml) (XMG1.2, BioXCell), mIL-6 (30 ng/ml) (Miltenyi Biotec) and hTGF-β1 (3 ng/ml) (StemCell). The following day, cells were treated with either DMSO or of FGIN-1-27 (10 μM). For phospho-ZAP70 analyses, cells were stained after 24 hours of treatment using 10 μg/ml of biotinylated anti-CD3 (145-2C11, BD Biosciences) and anti-CD28 (37.51, BD Biosciences) antibodies on ice for 15 minutes, washed, and stimulated in presence of 20 μg/ml of Streptavidin for 10 minutes at 37 degrees. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho ZAP70 antibody (n3kobu5, eBioscience) and anti-CD4 (RM4–5, eBioscience). For phospho-STAT3 analyses, cells were cultured for additional 24 hours in IL-6 free media, before stimulation with 100 ng/ml of mIL-6 for 30 minutes. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho-STAT3 antibody (4/P-STAT3, BD Biosciences) and anti-CD4. Dead cells were excluded using Live/Dead Amcyan (Thermo Fisher) staining prior to stimulation.

Cells were stained with mouse CD11c (N418, 1/200 and eBioscience), F4/80 (BM8, 1/200, Biolegend), or TCR-β (H57-597, 1/200, Biolegend) antibodies at 37 °C for 10 min, stimulated with cytokines for 15 min, fixed with 2% paraformaldehyde at 37 °C for 10 min, permeabilized in 90% ice-cold methanol for 30 min, washed twice with FACS buffer (PBS with 1% FBS), stained with pSTAT3 (Y705)-PE or pSTAT3 (Y705)-Alexa Fluor 647 antibodies (4/P-STAT3, 1/25 and BD Biosciences) at RT for 30 min, and analysed on a FACSCanto II (Becton Dickinson).