4.6. Plasmid, siRNA and transfection experiments

Plasmid RFP-GFP-LC3 was a gift from Professor Tamotsu Yoshimori (Osaka University, Osaka, Japan). The Cas9 expression plasmid pST1374-Cas9-N-NLS-Flag linker, the single guide RNA (sgRNA) plasmid pGL3-U6-sgRNA-PGKPuro and plasmid pPBB-EF-EGFP-C were gifts from Professor Ying Xu (Cambridge Suda Genome Resource Center, Soochow University, Suzhou, China). Plasmid pCMV-HA, pcDNA3.1-Flag and pcDNA3.1-Flag-AKT were purchased from Public Protein/Plasmid Library (PPL). A fragment of His-NLRP6 was cloned into pCMV-HA or pPBB-EF-EGFP-C. THP-1 macrophages were transiently transfected with plasmid using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h according to the manufacturer's instructions. HeLa cells were transiently transfected with plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) for 36 h according to the manufacturer's instructions. The negative control siRNA (NC siRNA) and siRNA specific for Fpn, Arnt, Mtf1 or Nrf2 were purchased from GenePharma. BMDMs and MEFs were transfected with 50 nM siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) for 48 h according to the manufacturer's instructions. Knockdown efficiency was assessed by quantitative PCR.