Sinrf2

Nrf2 siRNA and a scramble non-targeting siRNA (siCtrl; negative control) were purchased from Biomics Biotechnologies Co., Ltd. (Nantong, China). The siRNA sequences were as follows: siNrf2, 5′-GAGACUACCAUGGUUCCAA-3′; and siCtrl, 5′-UUCUCCGAACGUGUCACGU-3′. The siRNAs were transfected into cells using Lipofectamine 2000^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

ACHN cells were seeded in a 6-well plate (1×10⁵) for 24 h prior to transfection. Small interfering (si)Keap1, siNrf2, overexpressing Keap1, overexpressing Nrf2 and empty control plasmids were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). The sequence of siKeap1 was 5′-GAA TGA TCA CAG CAA TGA A-3′; the sequence of siNrf2 was 5′-GGT TGC CCA CAT TCC CAA ATC-3′; the sequence for overexpressing Keap1 was 5′-ATA CTC GAG ATG CAG CCA GAT CCC AGG CC-3′; the sequence for overexpressing Nrf2 was 5′-GTA CTA GTA TGA TGG ACT TGG AGT T-3′; and the NC sequence was 5′-GTT CTC CGA ACG TGT CAC GT-3′. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to perform the transient transfection, according to the manufacturer′s protocol. In total, 2 µg si/overexpressing RNA, negative control (NC) and Lipofectamine^® 2000 were added to Opti-Minimum Essential Medium (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 25°C for 20 min. Lipofectamine^® 2000 was subsequently mixed into each well, which was cultured in Opti-MEM RPMI 1640. After 6 h of culturing, the fluid was replaced with RPMI 1640 medium containing 10% FBS.