Peg400

The homodimer system (Takara, Japan) containing the chemical inducer of dimerization (CID, also called AP20187 or B/B Homodimerizer) and the pHom-Mem1 plasmid were purchased [18 (link)]. The intracellular domain of EpoR cDNA (247–406 amino acids) [23 (link)] was fused to GFP cDNA (pEGFP-N1; Takara), and the EpoR-GFP cDNA was inserted into the pHom-Mem1 plasmid containing a myristoylation signal peptide and the cDNA encoding the CID-binding protein (DmrB). The resulting product was ligated to a mouse Gata1 genomic fragment (GIHRD) spanning from 3.9 kb upstream of the first exon (IE exon) to the second exon [21 (link)]. The transgenic construct was referred to as G1HRD-idEpoRic (inducible dimerization of EpoR intracellular domain) and injected into fertilized eggs from BDF1 parents (CREA Japan). All mice were genotyped by PCR amplification of the GFP gene [25 (link)]. CID (10 mg/kg) and rHuEPO (300 U/kg; Chugai Pharmaceutical, Japan) were administered intraperitoneally. For oral administration, CID (100 mg/kg) was diluted with 50% PEG400 (Wako, Japan) in saline and immediately administered using a gastric tube. More than 3 mice or embryos in each experimental group were used in every experiment.

Colitis was induced in female mice by feeding 2.5% (for SCID mice) or 4% (for C57BL/6J and GFP-LC3#53 mice) DSS (mol wt 5000; Wako Pure Chemical Co., Osaka, Japan) dissolved in drinking water (distilled water). Mice were followed up until day 10, as described previously [74 (link)]. We used 2.5% DSS to induce colitis in C57BL/6J SCID mice because of its high lethality rate in VAD SCID mice. VAD B6 mice were intraperitoneally administered 50 mg/head/day of mAB against IL-1β (Biolegend, San Diego, CA, USA) from day 0 (when DSS treatment was initiated) to day 3. Isotype-matched IgG was injected as a control [75 (link)]. A chemical NLRP3 inhibitor, MCC950 (MedChem Express, Monmouth Junction, NJ, USA), was intraperitoneally administered to mice (10 mg/kg) at day 0 and every two days thereafter [76 (link)]. An autophagy activator (a potent and specific mTOR inhibitor), rapamycin (MedChem Express), was dissolved in ethanol and diluted ≥20-fold with 5% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) solution containing 5% PEG-400 (Wako Pure Chemical Co.). The drug was administered intraperitoneally to mice (10 mg/kg) daily from days 0 to 7 [77 (link)]. The inhibitors, antagonists, or antibody treatments were co-administered with DSS. We observed no interactions between DSS treatment and other drugs.

The following drugs were used: DZP (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan), PEG 400 (FUJIFILM Wako Pure Chemical), DMSO (Sigma-Aldrich), and Lucifer yellow (Sigma-Aldrich).