FIGURE 4.

Notch 1 regulates RORγt promoter activity. (A) In-vitro ILCHO treatment down-regulates RORγt and IL-17 mRNA expression. Total RNA was isolated from CD4⁺ T cells pretreated with 25μM ILCHO or DMSO as a vehicle control and cultured in Th17 polarizing conditions and analyzed by quantitative real time PCR. (B) Human naïve CD4⁺ T cells (1×10⁷) were nucleoporated with Notch 1 specific siRNA or scrambled siRNA followed by in-vitro Th17 polarization. Cells were harvested and RORγt expression was determined by quantitative RT-PCR. Transcript abundance was normalized to 18s rRNA expression. (C) Schematic representation of putative CSL binding sites in human RORγt promoter. (D) Specific primers were used to amplify putative CSL binding sites on human RORγt promoter. ChIP assay was performed to determine recruitment of Notch1 on RORγt promoter. Data shown represents fold recruitment of Notch1 on CSL binding sites on human RORγt promoter with respect to control IgG normalized with 1% input DNA. Semiquantitative PCR was also performed using 2μl of DNA eluates using specific primers against CSL sites in RORγt promoter to confirm transcript size. Data represents mean± SD of three independent experiments done in triplicates. * p≤0.05, **p≤0.01.