Lipofectamine 3000 transfection reagent

Human breast cell lines MCF-7, SKBR3, and MDA-231 (ATCC, U.S.A.) were used in our study. MCF-7 and MDA231 cells were maintained in DMEM medium, and SKBR3 cells were maintained in RMPI1640 medium. All media were supplemented with 10% fetal bovine serum (Gibco, U.S.A.) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). Along the culture, cells were kept in a humid air at 37°C with the percentage of CO₂ at 5%. In order to knock down HOTAIR, cells were transfected with HOTAIR shRNA (Genemediate Biological Tech., China) by using a Lipofectamine 3000 transfection reagent (Invivogen, U.S.A.) according to the manufacturer’s instructions.

Ultrapure lipopolysaccharide (LPS) (E. coli 0111:B4, cat. no. tlrl-3pelps), standard LPS (E. coli 0111:B4, cat. no. tlrl-eblps), ATP (cat. no. tlrl-atpl), nigericin (cat. no. tlrl-nig), and MSU (cat. no. tlrl-msu) were purchased from InvivoGen, lipofectamine 3,000 transfection reagent (cat. no. L3000015) was purchased from Thermo Fisher, C646 (S7152) was bought from Selleck Chemicals, mouse immunoglobin IgG protein (cat. no. ab198772) was purchased from Abcam, Protein A/G PLUS-Agarose (cat. no. sc-2003) was obtained from Santa Cruz, cell lysis buffer (CLB) (cat. no. 9803) was purchased from Cell Signaling Technology, and mouse IL-1β (cat. no. 88–7013), tumor necrosis factor-α (TNF-α) (cat. no. 88-7324), interleukin-6 (IL-6) (cat. no. 88-701364), and human IL-1β (cat. no. BMS22) ELISA kits were purchased from Thermo Fisher.
Anti–IL-1β (1:1,000, AF-401-NA; RRID:AB_416,684) was purchased from RD System, anti-NLRP3 (1:1,000, Cryo-2) and ASC (1:1,000, AL177) were purchased from Adipogen, anti–caspase-1 (1:1,000, ab179515), and anti-NEK7 (1:5,000, ab133514) were purchased from Abcam; Anti–β-actin (1:10,000, BH10D10), anti-p65 antibody (1:1,000, 8242), anti–p-p65 antibody (1:1,000, 3033), and GAPDH antibody (1:2,000, 5,174) were purchased from Cell Signaling Technology, and DyLight 488–labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen.