Accell sirna oligos

Transfections with siRNA oligos were performed using the Lipofectamin RNAiMax (Thermo Fisher Scientific). Briefly, human cells at density of 2.0x10⁵ cells/well were transfected in a 6-well plate with 40 pmol siRNA. Mouse cells at density of 3.0x10⁵ cells/well were transfected with 150 pmol siRNA. 24h after transfection the medium was exchanged. 72h post-transfection the cells were harvested and the levels of proteins of interest were assessed by immunoblot analyses as detailed in text. For silencing experiments in human and mouse cells pre-designed SMARTpool ON-TARGETplus and Accell siRNA oligos (Dharmacon; GE Healthcare) were used, respectively. For siRNA oligonucleotides details see Supplementary Table 2.

Purified mouse CD4⁺CD62L⁺ T cells or splenocytes were polarized under Th17 conditions with anti-CD3/CD28 Abs, or IRBP161-180, in the presence of Accell siRNA oligos (Dharmacon, Lafayette, CO) or random control siRNA in medium containing 3 % FCS as described previously (Chong et al., 2014 (link)). Cells were harvested on day 3 for follow-up experiments, or on days 4 and 7 to examine the efficiency and duration of IL-24 gene expression knockdown.