Hygrogold

HeLa tet-off cell lines (Clontech), in which expression from pTRE vectors is regulated by doxycycline, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; J R Scientific, Woodland, CA), 200 μg/ml G418 (Nacalai Tesque, Kyoto, Japan), 100 units/ml penicillin G (Sigma-Aldrich), and 100 μg/ml streptomycin (Sigma-Aldrich) in a humidified 5% CO₂/95% air atmosphere at 37°C. HeLa tet-off cell lines for doxycycline-regulated expression of YFP, SOD1-WT-YFP, or SOD1-G85R-YFP were selected in medium supplemented with 500 μg/ml HygroGold (Invivo-Gen, San Diego, CA) and 1.0 μg/ml doxycycline (Sigma-Aldrich). For transfections, cells were seeded on 3.5-cm dishes (BD, Franklin Lakes, NJ) or 3.5-cm glass-based dishes (Asahi-Technoglass, Tokyo, Japan) 1 day before transfection. All constructs were transfected using Effectene (Qiagen, Dusseldorf, Germany). For photoactivation analysis, mixtures of pTRE-SOD1-G85R-mPAGFP and pTRE-SOD1-G85R-TagRFP constructs (1:1) were transfected into HeLa tet-off cells. For FRET-FLIM assays, pTRE-SOD1-G85R-cp173mVenus or the control pTRE-SOD1-G85R-mVenus was cotransfected with pTRE-SOD1-G85R-mTFP1 at a ratio of 3:1.

Human THP-1 cells from the American Type Culture Collection (ATCC, Manassas, VA) were grown at 37°C with 5% CO₂ in RPMI-1640 medium supplemented with 10% FBS (v/v), 100 U/ml-100 µg/ml penicillin-streptomycin, and 50 µM β-mercaptoethanol. The NLRP3-deficient THP-1 cells (THP1-defNLRP3, InvivoGen) were grown at 37°C with 5% CO₂ following InvivoGen’s instruction in RPMI-1640 medium supplemented with 10% FBS (v/v), 200 µg/ml HygroGold, and 100 µg/ml normocin. HEK-Blue IL-1β cells that contain an IL-1β-sensitive reporter were from InvivoGen and grown following InvivoGen’s instruction at 37°C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS (v/v), 4.5 g/l glucose, 2 mM GlutaMAX medium, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml zeocin, 200 µg/ml hygromycin, and 100 µg/ml normocin (all from Life Technologies or InvivoGen).The level of secreted embryonic alkaline phosphatase (SEAP) protein, a truncated form of human placental alkaline phosphatase released into the culture medium, was used as a measurement of NF-κB activation through IL-1β stimulation with the QUANTI-Blue™ detection reagent.