Characterization of Aire in CB-SC. (A) Expression of Aire mRNA in CB-SCs. Real time PCR analysis for Aire mRNA expression followed by electrophoresis in 2% agarose gel. Data are representative of three CB-SC preparations. (B) Immunocytochemistry for Aire. Isotype-matched IgG served as control (left) for Aire staining (right) with magnification ×200. (C) Western blot shows the dose-dependent knockdown response of Aire following siRNA treatment. (D) Effects of Aire knockdown on PD-L1. Western blot demonstrates decreased expression of program death ligand-1 (PD-L1) in CB-SC following knockdown of Aire expression by siRNA. CB-SC cells transfected with negative control siRNA (NC siRNA) served as control for three pairs of human Aire-specific siRNA (P1, P2, and P3) at optimal concentration (50 nM). Representative data of those obtained from five experiments. (E) Effects of Aire knockdown on co-cultured lymphocytes. Flow analysis of Treg population following culture of lymphocytes alone, in the presence of phytohaemagglutinin (PHA, 10 μg/ml), in the presence of PHA and NC siRNA-treated CB-SCs, and in the presence of PHA and Aire siRNA (50 nM)-treated CB-SCs. Representative data obtained from three experiments. Aire, autoimmune regulator; CB-SCs, cord blood stem cells; IgG, immunoglobulin G: PCR, polymerase chain reaction; PHA, phytohaemagglutinin; siRNA, small interfering RNA; T1D, type 1 diabetes; Tregs, regulatory T cells.