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. 2014 Dec 22;35(2):344–355. doi: 10.1128/MCB.00926-14

FIG 2.

FIG 2

Knockdown of Dgcr14 mRNA suppressed the function of RORγt. (A) The left panel shows RT-qPCR for assessment of Dgcr14 in 68-41 cells transfected with control or Dgcr14 RNAi-expressing vectors. The mRNA levels of all genes were normalized to the level of Gapdh mRNA expression. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05. The right panels show Western blotting for DGCR14 proteins in 68-41 cells transfected with control or Dgcr14 RNAi-expressing vectors. (B) Luciferase reporter assay of 68-41 cells. After transfection with Dgcr14 RNAi expression vectors, an mIl17a promoter luciferase vector, a β-gal expression vector, and/or a RORγt expression vector, cells were cultured for 1 day. Cells were then harvested, and luciferase assays were performed. As an internal control, ONPG-dependent β-gal activities were measured with a microplate reader. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05. (C) RT-qPCR analysis of Dgcr14 RNAi-expressing 68-41 cells. After transfection with or without Dgcr14 RNAi expression vectors, 68-41 cells were cultured with or without anti-CD3ε antibody, IL-6, and TGF-β. RNA was extracted and RT-qPCR was performed. The mRNA levels of all genes were normalized to the level of Gapdh mRNA expression. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05. (D) The left panel shows RT-qPCR assessment for Dgcr14 mRNA in 68-41 cells transduced with control (sh-luc)- or two Dgcr14 shRNA-expressing retroviruses (sh-Dgcr14#1 and sh-Dgcr14#2). The mRNA levels of all genes were normalized to the level of Gapdh mRNA expression. After transduction with each virus, GFP-positive cells were isolated with a FACSAria (BD) and cultured. Among six shRNAs for Dgcr14, two shRNAs targeting Dgcr14 reduced its mRNA levels. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05. The right panels show Western blotting for DGCR14 in 68-41 cells transduced with control or Dgcr14 shRNA-expressing retroviruses. Western blot assays were performed with the antibodies indicated. (E) RT-qPCR assessment of Il17a mRNA in 68-41 cells transduced with control or Dgcr14 shRNA-expressing retroviruses. The mRNA levels of all genes were normalized to the level of Gapdh mRNA expression. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05.