Si nrf2

Hemin and Mel (Aladdin, China) were dissolved in absolute ethyl alcohol and diluted with 0.9% normal saline. Ro 31 were purchased from TargetMol, United States. Luz were purchased from Santa Cruz Biotechnology, United States. We transfected astrocytes with Nrf2 specific small interfering RNA (si-Nrf2) (GenePharma, China) by Lipofectamine^® 2000 transfection reagent (Invitrogen, United States) according to the manufacturer’s instructions. Western blotting was applied to prove the si-Nrf2 knockdown efficiency.

The small interference RNA (siRNA)-targeting bovine Nrf2 gene (si-Nrf2) and a negative control siRNA (si-NC) were synthesized by GenePharma. The sense sequence of si-Nrf2 is 5′-CAGUUGAGGACUUCAAUGAdTdT-3′, and the antisense sequence is 5′-UCAUUGAAGUCCUCAACUGdTdT-3′) [45 (link)]; the sense sequence of si-NC is 5′-UUCUCCGAACGUGUCACGUdTdT, and the antisense sequence is 5′- ACGUGACACGUUCGGAGAAdTdT-3′. Transfection was performed using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After 12 h of transfection, cells were treated as described above; the MAC-T cells were harvested 48 h after transfection to detect Nrf2 mRNA levels.

The human NSCLC cells cell lines HCC827, H460, and A549 were purchased from American Type Culture Collection (ATCC, USA). These cells were cultured in RPMI1640 (Welgene, Daegu, South Korea) supplemented with 10% foetal bovine serum (FBS) (Welgene, Daegu, South Korea) and 1% penicillin/streptomycin at 37 ℃ in a humidified atmosphere containing 5% CO₂. Brusatol (Carbosynth, UK) was purchased and dissolved in dimethyl sulfoxide (DMSO) (D2650, Sigma Aldrich, USA). MG132 was purchased from CalBiochem (SanDiego, CA, USA). Nrf2-EGFP and Keap1-Flag vectors were purchased from Addgene (http://www.addgene.org). The oligo ribonucleotide sequences of human Nrf2 siRNA (siNrf2) were as follows: 5′-UCUGACUCCGGCAUUUCACUTT-3′ (sense) and 5′-AGUGAAAUGCCGGAGUCAGATT-3′ (antisense) (Shanghai GenePharma Co. Ltd., China).

To silence the gene expression of Nrf2, Nrf2-specific siRNA (SiNrf2) and control siRNA (SiNC) were obtained from Genepharma (Shanghai, China). HK-2 cells were seeded into 6-well plates (2 × 10⁵ cells per well) overnight and then transfected with siRNA (SiNrf2 or SiNC) mixed with Lipofectamine 2000 transfection reagent. The medium (Opti-MEM) was replaced at 6 h post-transfection. After 24 h, the cells were treated with DDP and CNPs. SiNrf2 sequences were enlisted in Table S1.

A total of 2 × 10⁶ Marc-145 cells were transfected into 24-well plates with 50 pmol of siNC (negative control) and siNrf2 (GenePharma, Shanghai, China) using 5 µL of Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). After 24 h, the cells were treated with Xn and infected with PRRSV (0.01 MOI) for 36 h. Cells were harvested for Western blotting and qRT-PCR. The sequences of siRNAs targeting Nrf2 were: (a) 5′-AGA CAA ACA TTC AAG CCG-3′; (b) 5′-AGA ATA AAG TGG CTG CTC-3′.