Hek 293t

The human HCC cell lines PLC, Hep3B, SMMC-7721, Huh7, HepG2, the normal liver cell line L02 and embryonic kidney cell line HEK-293T were purchased from the Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Science (Shanghai, China). Human HCC cell lines MHCC-97H and HCC-LM3 were established at the Liver Cancer Institute, Zhongshan Hospital, Fudan University. These cells were stored in liquid nitrogen and cultured in 5% CO2 at 37°C with high glucose Dulbecco's modified Eagle media (GIBCO, Grand Island, NY) supplemented with 10% FBS (GIBCO).
The negative control small interference RNA (siNC, 5′-UUCUCCGAACGUGUCACGUTT-3′), and Nrf2 siRNA (5′-CAUUGAUGUUUCUGAUCUATT-3′) were synthesized and inserted into the LV3-GFP vector (named as LV3-shCon or LV3-shNrf2). For Nrf2 overexpression, the Nrf2 expression plasmid was cloned into LV5-GFP vector (named as LV5-Nrf2). Thereafter, HEK-293T cells (1 × 10⁶ cells) were transfected with the respective plasmid (LV3-shCon, or LV3-shNrf2, or LV5-NC, or LV5-Nrf2) and packaging vectors by GenePharma Co. Ltd (Shanghai,China). HCC cells were then infected with the packaged lentivirus under medium culture.

The HEK293T cell line and the human T‐ALL cell line (Jurkat) were purchased from ATCC (Manassas, VA, USA). Jurkat cells were grown in the RPMI1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin, while HEK293T in the 10% FBS and 1% penicillin/streptomycin‐contained DMEM (HyClone, Logan, UT, USA). All cells were cultured at 37°C in a 5% CO₂ humidified atmosphere. When the cell density reached 80%, the cells were transfected as per the protocols of the lipofectamin 2000 kit (11668‐019, Invitrogen, New York, California, USA). After 48 hours transfection, the cells were then cultured in the 10% FBS‐contained medium for 24‐48 hours. miR‐204 mimic and miR‐204 inhibitor were purchased from GenePharma (Shanghai, China).
HEK293T cells were transfected with (1) mimic‐NC + WT‐IRAK1, (2) mimic‐miR‐204 + WT‐IRAK1, (3) mimic‐NC + Mut‐IRAK1 and (4) mimic‐miR‐204 + Mut‐IRAK1; Jurkat cells were transfected with (1) mimic‐NC, (2) mimic‐miR‐204, (3) inhibitor‐NC, (4) inhibitor‐miR‐204, (5) mimic‐NC + oe‐NC, (6) mimic‐miR‐204 + oe‐NC, (7) mimic‐miR‐204 + oe‐IRAK1, (8) mimic‐NC + oe‐NC, (9) mimic‐miR‐204 + oe‐NC, (10) mimic‐miR‐204 + oe‐p65, (11) oe‐NC + si‐NC, (12) oe‐IRAK1 + si‐NC or (13) oe‐IRAK1 + si‐p65.