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. Author manuscript; available in PMC: 2015 Oct 3.
Published in final edited form as: Biochem Biophys Res Commun. 2014 Sep 18;452(4):1040–1045. doi: 10.1016/j.bbrc.2014.09.037

Figure 3. Specific silencing of RORγt in human CD4+ T cells by CD4-AshR-RORγt chimera.

Figure 3

Karpas 299 cells and PBMCs were treated as described in methods. (A through C) Quantitative real-time PCR assay for RORγt gene expression. RORγt gene expression was significantly reduced by CD4-AshR-RORγt chimera in a concentration-dependent manner in Kapas 299 cells (A) and T-cell enriched PBMCs (B). Mock-CD4-AshR-RORγt chimera, CD4-AshR-scrambled control or CD4-AshR-CCR5 chimera had no effect on RORγt gene expression in T-cell enriched PBMCs (C). Additionally, all the chimeras lacked a significant inhibition on TBX21or GATA3 in T-cell enriched PBMCs (C). (Data are presented as mean ± SD of three experiments). 1, PBS; 2, CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4, CD4-AshR-scrambled control chimera; 5, CD4-AshR-CCR5 chimera. (D and E) RORγt protein expression was analysed by flow cytometry. (D) Karpas 299 cells were stimulated with PMA 50 ng/ml for 24 h. Red line, CD4-AshR-RORγt chimera; Blue line, Mock-CD4-AshR-RORγt chimera (representative of three experiments). (E) PBMCs were stimulated with anti-CD3/CD28 and LPS for 48 h. RORγt expression was reduced by CD4-AshR-RORγt chimera (red line), but not by mock-CD4-AshR-RORγt chimeras (blue line) or CD4-AshR-scrambled control chimera (purple line). Black line, PBMCs without stimulation; green line, PBMCs with stimulation but without chimeras (representative of three experiments). (F) The percentage of RORγt+ cells in stimulated T-cell enriched PBMCs was reduced by CD4-AshR-RORγt chimeras, but not by mock-CD4-AshR-RORγt chimera or CD4-AshR-scrambled control chimera. 1, PBMCs without stimulation; 2, PBMCs with stimulation but without chimeras; 3, Stimulated PBMCs were treated with CD4-AshR-RORγt chimeras; 4, Stimulated PBMCs were treated with mock-CD4-AshR-RORγt chimera; 5, Stimulated PBMCs were treated with CD4-AshR-scrambled control chimera (Data are presented as mean ± SD of three experiments).