Bay 11 7821

For local treatment with chemicals or proteins, AG1X2-formate beads (for chemicals) or Affigel Blue beads (BIO-RAD, 1537302; for BMP4) were soaked in different concentrations of the desired protein or chemical overnight at 4 °C. Beads were washed in PCS before grafting. Dimethyl sulfoxide (DMSO, 0.2%) or BSA (0.1%) was used to dilute the chemicals or proteins, respectively, and for soaking the control beads. Final concentrations used for microbead-soaking: 50 ng/μl recombinant human BMP4 (R&D systems, 312-BP), 200 µM dorsomorphin dihydrochloride (Tocris, 3093), 2 µM ionomycin (Sigma, I9657). For chemical treatment to the whole embryo, the chemical was diluted first in PBS (1:10 v:v) and then in egg albumen (9:10 v:v), which was used to culture the embryos (under the vitelline membrane). For treatments with VIVIT (Tocris, 3930) and BAY 11-7821 (Tocris, 1744), embryos were first soaked in the chemical diluted in PCS for 1 h, prior to culture with albumen containing the same concentration of the chemical. Final concentrations used for treatment of whole embryos: 20 µM dorsomorphin, 200 µM flufenamic acid (Sigma, F9005), 2 µM ionomycin, 50 µM, nicardipine (Sigma, N7510), 20 µM U73122 (Sigma, U6756), 20 µM U73343 (Sigma, U6881), 12 µM VIVIT (Tocris, 3930), 12.5 µM BAY 11-7821 (Tocris, 1744).

Peptidoglycan (PGN) from S. aureus, mouse anti-human immunoglobulin E antibody (ε-chain specific) (anti-IgE) were purchased from Sigma. Human Myeloma IgE was from Merck. Pam3CSK4 was purchased from Invivogen. SP600125, SB203580, PD98059, ciclosporin and Bay11-7821 were from Tocris. Wortmannin was from Cayman. FITC-conjugated anti-human FcεRI antibody and FITC-conjugated mouse IgG2b isotype control were purchased from eBioscience. When chemicals were dissolved in DMSO, the final concentration of DMSO did not alter the normal response of LAD2 cells.

SUM190 and SUM149 were obtained from Asterand (Detroit, MI, USA) and cultured in Hams F-12 media (Mediatech, Manassas, VA, USA) containing 5 μg/ml insulin, 1 μg/ml hydrocortisone, antibiotics (penicillin/streptomycin), and 5% (SUM149) or 2% (SUM190) of fetal bovine serum (FBS) (HyClone, Logan, UT, USA). Medium for SUM190 cells was further supplemented with 5 mM ethanolamine, 10 mM HEPES, 5 μg/ml transferrin, 6.6 ng/ml 3,3',5-triiodo-L-thyronine sodium salt, 8.7 ng/ml sodium selenite, and 1 mg/ml bovine serum albumin (BSA). Cells were cultured at 37 °C in a 5% CO₂ incubator. Breast cancer cell lines BT483 and Cama-1 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM-F12 (1:1) supplemented with 10% FBS. Poly(I:C) was obtained from InvivoGen (San Diego, CA, USA). Doxorubicin hydrochloride, 5-Fluorouracil, Paclitaxel, insulin, hydrocortisone, HEPES, and BSA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cardamonin and Bay 11-7821 were purchased from TOCRIS Bioscience (Ellisville, MO, USA).