Liposomal transfection reagent

SI0211352 and S100186424 AIRE-specific siRNA (Qiagen, Hilden, Germany) were used for silencing AIRE gene expression in mTECs. The sequences of sense and antisense strands of AIRE-specific siRNA were respectively: 5′-GGAAGAUCCAAGAAGUGCATT-3′ and 5′-UGCACUUCUUGGAUCUUUCCTG-3′ for SI0211352; 5′-GUGGCAAUUUGAAGAACAATT3′ and 5′-UUGUUCUUCAAAUUGCCACTG-3′ for S100186424. mTECs (4×10⁵) were transfected with 2.5 μg of AIRE-specific siRNA using the liposomal transfection reagent (Roche, Mannheim, Germany). Cells were cultured for 24 h at 37°C in culture medium before extracting total RNA and quantifying AIRE mRNA expression by Real Time PCR.

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The cells were incubated in a thermostatic incubator at 37°C in an atmosphere with 5% CO₂. The cells were seeded on a 96-well plate at a density of 3,000 cells/well and were transfected with liposomal transfection reagent (Roche). At 24, 48 and 72 h after transfection, cell proliferation was analyzed using MTT (Sigma Aldrich, St. Louis, MO, USA). Briefly, 20 μl MTT was added to the 96-well plate and incubated at 37°C for 4 h until purple precipitate was visible. Then, the culture supernatant was discarded and 150 μl dimethylsulfoxide was added. The 96-well plate was oscillated for 10 min until the purple precipitate was dissolved. The absorbance was measured at 492 nm on a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Cell proliferation was calculated based on these absorbance values.