Accell delivery medium

N/TERT KCs were plated in 96-well plates (30,000 cells/well) and incubated at 37°C with 5% CO₂ overnight. Accell siRNA (100 μM; Dharmacon: APOBEC3A, E-017432-00-0005; APOBEC3B, E-017322-01-0005; APOBEC3G, E-013072-00-0005; APOBEC3H, E-019144-00-0005; DNASE1, E-016280-00-0005) was prepared in 1× siRNA buffer (Dharmacon, B-002000-UB-100). One microliter of 100 μM siRNA was diluted with 100 μL Accell delivery medium (Dharmacon, B-005000) for each well of 96-well plates. The growth medium was removed from the cells and 100 μL of the appropriate delivery mix with siRNA was added to each well and the plate was incubated at 37°C with 5% CO₂. Accell Non-targeting Control siRNA (Dharmacon, D-001910-01-05) was used as a negative control. After 72 hours, cells were harvested for RNA preparation. RNA isolation and qRT-PCR were as described above.

For small interfering RNA (siRNA) knockdown of TNFAIP3, Accell SMARTpool siRNA (Dharmacon, Colorado, United States) was used according to the manufacturer's instructions. Briefly, 1 × 10⁵_effTreg cells per well were stimulated with αCD3/CD28 beads (1:5 of bead:cell ratio) in Accell Delivery Medium (Dharmacon) supplemented with rhIL-2 (100 U/ml) and incubated with 1 mmol cyclophilin B siRNA (positive control), or 1 mmol non-targeting control siRNA, or 1 mmol TNFAIP3 siRNA for 120 h (for siRNA sequences, see Table S2). Quantitative real-time PCR (RT-qPCR) was performed to confirm the knockdown of the target gene expression.