File S1 - A Novel Small Compound SH-2251 Suppresses Th2 Cell-Dependent Airway Inflammation through Selective Modulation of Chromatin Status at the Il5 Gene Locus

The effects of SH-2251 on Th1-, Th9-, and Th17-differentiation. Naïve CD4 T cells were cultured under Th1- (A), Th9- (B) or Th17- (C) conditions in the presence or absence of SH-2251 (100 nM) for five days. The cells were restimulated with an immobilized anti-TCR-β mAb for six hours, and the intracellular staining profiles were determined using intracellular staining (left). The following antibodies were used for intracellular staining: anti-IL-4-PE mAb (11B11; BD Bioscience), IFN-γ-FITC mAb (XMG1.2; BD Bioscience), anti-IL-9-PE mAb (RM9A4; BioLegend), anti-IL-17A-Alexa647 mAb (TC11-18H10.1; BioLegend) and IL-17F-Alexa488 mAb (9D3.1C8; BioLegend). The percentages of each quadrant are indicated. The cytokine production by the SH-2251-treated Th cells stimulated with an immobilized anti-TCR-β mAb for 16 hours was determined with ELISA. The culture conditions for each Th cell differentiations were as follows. Th1-conditions: IL-2 (2.5 ng/ml), IL-12 (1 ng/ml; PeproTech) and anti-IL-4 mAb (5 µg/ml; 11B11; BioLegend). Th9-conditions: IL-2 (2.5 ng/ml), IL-4 (10 ng/ml), TGF-β (10 ng/ml; PeproTech) and anti-IFN-γ mAb (5 µg/ml). The Th17-conditions were as follows: IL-6 (10 ng/ml; PeproTech), IL-1β (5 ng/ml; PeproTech), TGF-β (1 ng/ml), anti-IL-2 (5 µg/ml; BioLegend), anti-IL-4 mAb (5 µg/ml) and anti-IFN-γ mAb. Three independent experiments were performed with similar results. *PPt-test). (DOCX)