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.
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.