Roswell park memorial institute (rpmi)

The human colorectal cancer cell lines SW480, SW620, Caco-2, HT-29 and DLD-1 were purchased from ATCC (American Type Culture Collection) and maintained in DMEM (Welgene, LM011–05) and RPMI (Welgene, LM011–01) supplemented with 10% FBS (Gibco, 16000–044) and 1% antibiotics (Gibco, 15240–062). All of the indicated cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO₂ (Heraeus BB15 CO2 Incubator, Thermo Fisher). All the cell lines used in this study were authenticated and validated using STR genotyping performed by HPBIO, Inc. (Seoul, South Korea). Results of STR genotyping were provided in supplemental Table 3.

MCF10A, ZR75-30, MDA-MB-453, BT20, JIMT-1, MDA-MB-231, and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF7, T47D, ZR75-1, SKBR3, BT474, HCC-1954, HCC1419, and HS578T cells were purchased from the Korean Cell Line Bank. MCF7, T47D, MDA-MB-231, and HEK293T cells were cultured in DMEM (Welgene, Daegu, Republic of Korea) supplemented with 10% FBS, and SKBR3, BT474, HCC-1954, HCC-1419, JIMT-1, and HS5788T cells were cultured in RPMI (Welgene) supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. Trastuzumab was obtained from Roche (Basel, Switzerland). The β-Catenin/TCF inhibitor FH-535 was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Testing bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, and a testing fungus, Candida albicans, were purchased from Korean Cell Line Bank (Jongno-gu, Seoul, Republic of Korea). BBL^TM Mueller Hinton Broth (Spectrum Chemical MFG Co., New Brunswick, NJ, USA) and DIFCO^TM Nutrient agar (BD, Becton Drive Franklin Lakes, NJ, USA) were used to cultivate bacteria and fungus and to perform antimicrobial susceptibility testing. To prepare the culture media, 11 g of Mueller–Hinton Broth and 11.5 g of Nutrient agar were individually dissolved in 500 mL of distilled water. After that, they were sterilized by autoclaving at 120 °C for 15 min. In the case of Nutrient agar, the sterilized medium solution was solidified in Petri dishes before use. A mouse L929 fibroblastic cell line was also purchased from Korean Cell Line Bank. L929 cells were grown in Roswell Park Memorial Institute medium (RPMI; Welgene, Daegu, Republic of Korea) containing 10% fetal bovine serum (FBS, Gibco^®, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin solution (Welgene) at 37 °C in a 5% CO₂ incubator.

THP-1 cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI (Welgene, Daegu, Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY) and 1% penicillin-streptomycin (PS; Lonza, Basel, Switzerland) at 37°C in 5% CO₂. THP-1 cells were differentiated into macrophages by treatment for 3 h with 300 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO). 24h later, the cells were stimulated to activate NLRP3 inflammasome.
For NLRP3 inflammasome stimulation, THP-1-differentiated macrophages were primed with 2 μg/mL LPS (Ultra-pure LPS, InvivoGen, San Diego, CA) for 4 h, and then treated with 5 mM ATP (InvivoGen) for 45 min. After the cells were washed with phosphate buffered solution (PBS) three times washing, the cells were cultured in high glucose DMEM (Welgene) with 2% (vol/vol) heat-inactivated FBS (Gibco) and 1% PS (Lonza) at 37°C in 5% CO₂ until further assays.
For treatment, rapamycin (1 to 1000 nM; Sigma-Aldrich), BafA1 (75 nM; Sigma-Aldrich), 3-MA (2.5 mM; Sigma-Aldrich), IL-1ra (1 to 200 ng/mL; Sigma-Aldrich), recombinant human IL-1β (1 to 1000 pg/mL; Sigma-Aldrich), or SB-208350 (1 to 100 μM; Sigma-Aldrich) were added to the cultures simultaneously with LPS priming step and maintained until assays.

The HCC cell lines ;SNU449, SNU475, SNU398, SNU898, Huh7, HepG2, Hep3B and PLC/PRF/5 were purchased from the Korean Cell Line Bank. Huh6 cells were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Germany). All HCC cells were maintained in RPMI (Welgene, Korea) or Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (p/s; Gibco, Grand Island, NY, USA). Fa2N-4, a human immortalized hepatocyte cell line, was obtained from Xenotech (Lenexa, KS, USA) and first cultured in serum-containing plating medium (K4000; Xenotech). Miha. Cells were kindly provided by Dr. Kim. K. M. from ASAN medical center. To produce tumor spheroids, HCC cells seeded at a density of 6 × 10³ cells/well in 96-well round-bottom ULA microplates (Corning Life Sciences, Corning, NY, USA). All cells were maintained in a 5% CO₂ humidified incubator at 37 °C.

The assay kit obtained from Sigma-Aldrich was used. SKBR3 (3 × 10³ cells/well), BT474 (5 × 10³ cells/well), JIMT-1 (5 × 10³ cells/well) and HCC-1954 (3 × 10³ cells/well) cells in RPMI (Welgene) containing 10% FBS were plated in a 96-well plate and incubated with trastuzumab for five days. After fixing in trichloroacetic acid (TCA) for an hour at 4 °C, cells were stained for 30 min with 0.4% sulforhodamine B, destained in 1% acetic acid, dissolved in 10 mM Tris, and examined at 490 nm.

Jurkat T cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI (Rosewell Park Memorial Institute) medium (Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/mL), penicillin G (100 units/mL) and l-glutamine (2 mM). Cells were grown at 37 °C in a humidified incubator containing 5% CO₂ and 95% air.