Interleukin 3 (il 3)

For scRNA-seq in Fig. 1, BMBAs generated by culturing bone marrow cells in the presence of IL-3 (0.3 ng/mL: BioLegend) for 7 days were subjected to scRNA-seq analysis. BMBAs generated from 5 mice were pooled to generate scRNA-seq data. For scRNA-seq in Fig. 2, single-cell samples were prepared from the bone marrow and spleen of Mcpt8^GFP transgenic mice and depleted of lineage-positive cells by using biotinylated lineage antibodies (anti-CD4, anti-CD8a, anti-CD19, anti-B220, anti-Gr-1 and anti-TER-119, dilution 1:400) and EasySep Mouse Streptavidin RapidSpheres Isolation Kit (Stemcell Technologies, catalog #: ST-19860). GFP-positive cells were sort-purified from lineage-negative cells. Basophils isolated from 5 mice were pooled to generate scRNA-seq data. For scRNA-seq in Fig. 5, single-cell suspensions were prepared from the bone marrow and infected skin and subjected to positive selection of CD49b-positive cells by using biotinylated anti-CD49b antibody (BioLegend; clone: DX5, catalog #: 108904, dilution 1:400) and EasySep mouse Biotin Positive Selection Kit II (Stemcell Technologies, catalog #: ST-17665) followed by the isolation of CD200R3⁺cKit^- cells by cell sorting. Basophils isolated from 5 mice were pooled to generate scRNA-seq data.

After the primary separating of bone marrow cells from wild-type mouse, IL-3 and SCF (Biolegend, California, USA)-induced differentiation was used to get purified mast cells. Then the flow cytometry analysis was used to separate CD117 and FcεRIα positive mast cells (APC-CD117 and FITC-FcεRIα, Biolegend, California, USA). Serum containing IgE from OVA-induced mice was used to activate purified mast cells overnight. Mast cells were challenged with 200μg OVA for 1h, 3h and 6h separately. Then, Real-time PCR was used to detect the mRNA expression level of Gpr97 on mast cells.

Murine macrophage cells (RAW 264.7) were obtained from the American Type Culture Collection (ATCC Accession number TIB-71). Dulbecco's modified Eagle's medium (DMEM), Minimum Essential Medium Eagle (α-MEM), and Fetal Bovine Serum (FBS) were purchased from Invitrogen-Gibco (Carlsbad, CA, USA). The mouse IL-3 (Cat number 432102, Lot: B154176), IL-4 (Cat number 431102, Lot: B156652), IL-6 (Cat number 431305, Lot: B165924), IL-1α (Cat number 433402, Lot: B171801), TNF (Cat number 430902, Lot: B170648), and enzyme-linked immunosorbent assay (ELISA) kit were purchased from BioLegend's ELISA MAX (San Diego, CA, USA). LPS derived from P. gingivalis (Pg) was purchased from Cedarlane (Ontario, Canada). For cells viability assay, alamarBlue was purchased from Invitrogen (Cat number DAL1025) (Grand Island, NY, USA) and protein concentrations were determined by Pierce BCA from Thermo Scientific (Cat number 23225) (Rockford, IL, USA). Mouse-derived RANKL, dimethyl sulfoxide (DMSO) ≥99.5%, penicillin G-streptomycin, acid phosphatase kits for tartrate-resistant acid phosphatase (TRAP) staining, 4,6-diamidino-2-phenylindole (DAPI) for DAPI staining, quercetin 98% (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one,3,3′,4′,5,6-entahydroxyflavone), and the other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).

HAP1 cells were cultured in IMDM (Nacalai Tesque) with 10% Fetal Bovine Serum (FBS). HEK293T or U2OS cells were cultured in DMEM-high glucose (Nacalai Tesque) supplemented with 10% FBS. Ba/F3 cells were cultured in IMDM with 10% FBS and interleukin-3 (IL-3, 1 μg/mL, Biolegend). hTert1-RPE1 were maintained in DMEM:F12 supplemented with 10% FBS and hygromycin 0.01 mg/ml. HL60 were cultured in RPMI1640 supplemented with 10% FBS.

Colony assays in liquid medium were performed as described previously^{18 (link)}. Erythroid colony assays were performed on DsRed⁺CD13^low sorted HSPC on day 3 of culture by limiting dilution in 96-U plates (0.3 cells/well) in Iscove's Modified Dulbecco's Medium (IMDM; Fisher Scientific, Loughborough, UK) supplemented with IL-3, IL-6, SCF and EPO (BioLegend, London, UK) at 5 ng/mL or 2 U/mL for EPO and incubated at 37 °C with 5% CO₂. Following 7 days of growth, individual erythroid colonies (> 50 cells) and clusters (> 5, < 50 cells) were counted and scored. BFU-e and CFU-e were not discriminated in these counts. To assess their self-renewal potential, colonies were harvested, replated at higher density (1 cell/well), and cultured for an additional week.