FIG. 4.

AIRE activates the transcription of Ins and Spt1 genes in mTECs. (A) AIRE activates transcription from the human Ins promoter. 1C6 cells coexpressed AIRE and the plasmid target containing the human Ins promoter linked to the firefly luciferase gene, and luciferase activity was measured. Renilla luciferase readings were used to normalize the firefly luciferase activity of each sample for all transfections. The expression of AIRE was confirmed by Western blotting and is presented below the bar graph. Error bars denote standard errors of the means of three independent transfections. (B) AIRE activates expression of Ins2 and Spt1 genes in mTECs. RNA was extracted from 1C6 cells transiently expressing AIRE (lanes 1 to 4) or the empty plasmid vector (lanes 5 to 8) and treated additionally with 100 mM TSA for 12 h or from AIRE.1C6 cells that stably expressed AIRE (lane 9). Semiquantitative RT-PCR (fourfold serial dilution) analyses of several tissue-specific genes were performed. PCR was carried out using gene-specific primers as indicated. (C) AIRE mRNA levels in mTECs. RNA was isolated from 1C6 cells (lane 1), primary (1°) mTECs (lane 2), and transiently (lane 3) or stably (lane 4) transfected 1C6 cells, and RT-qPCR was performed with primers specific for AIRE. mRNA levels were normalized to actin.