Z02922 10

The cells were seeded in 96-well culture plates at appropriate density were first primed with LPS (100 ng/mL) (00497693, Thermo Fisher Scientific) for 4 h, followed by treatment of 3% DSS for 24 h prior to treatment for 48 h with ERB-041 (1 mM) and TNF-a (5 ng/mL) (Z01001-10, Genscript) or IL-1b (10 ng/mL) (Z02922-10, Genscript). The cell activity was tested according to CCK-8 (40203ES76, YEASEN) protocol. The absorbance at 450 nm was obtained by a microplate reader with Gen5 CHS 2.07 software (BioTek, USA).
Generation of knockdown stable cells pLVX-shRNA2 (PT4052-5, Clontech), psPAX2 (PVT2320, Life Science Market) and pMD2.G (PVT2321, Life Science Market) vectors were purchased from Nova Lifetech Limited (Hongkong, China). Lentivirus-mediated shRNA targeting NLRP6 and ERb mRNA sequences (Listed in Table S1) were obtained from Genscript Biotech Corporation.
pLVX-shNLRP6/pLVX-shERb, psPAX2, and pMD2.G were transfected into the HEK239T cells. The supernatant containing lentivirus was harvested after 40 h and then added into the NCM-460 cells. After 24 h, the medium was replaced with fresh medium. 48 h later, cells were cultured in selection medium containing 10 mg/mL puromycin. All these stable transfected cells were tested regularly by western blotting analysis to ensure the efficiency of down-regulation.

The cells were seeded in a 6-well plate at an appropriate density. After LPS priming and 3%DSS, ERB-041 (1mM) and TNF-a (5 ng/mL) (Z01001-10, Genscript) or IL-1b (10 ng/mL) (Z02922-10, Genscript) stimulation, the cells were loaded with 4 mM of MitoSOX (M36008, Thermo Fisher Scientific) for 20 min. Fluorescence intensity was determined using Flow Cytometer (Beckman, USA) with Accuri C6 software. FlowJo 10.0 FACS software was used for the data analysis.

The cells were seeded in 22 mm confocal dishes at an appropriate density. After LPS priming and 3% DSS, ERB-041 (1 mM) and TNFa (5 ng/mL) (Z01001-10, Genscript) or IL-1b (10 ng/mL) (Z02922-10, Genscript) stimulation, the cells were incubated for 30 min at 37 C with 250 nM MitoTracker Red CMXRos Mitochondrial Probe (M7512, Thermo Fisher Scientific). The fluorescence signals were measured by Zeiss microscope (excitation at 579 nm; emission at 599 nm).