Heregulin β1 hrgβ1

Retro-2 (2-([(5-Methyl-2-thienyl)methylene]amino)-N-phenylbenzamide; C₁₉H₁₆N₂OS; R2) was purchased from EMD Millipore (Temecula, CA, USA) and stored at 50 mg/mL in DMSO at −20 °C. R2 spontaneously cyclizes to Retro-2^cycl [52 (link)], the mixture is referred to as R2 herein. The purified Retro-2.1 (6-Fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one; C₂₃H₁₈FN₃OS₂; R2.1) S-enantiomer ((S)-R2.1) and R-enantiomer ((R)-R2.1) were purchased from ChiroBlock (Wolfen, Germany). Heregulin β1 (HRGβ1) was purchased from R&D Systems Inc (Minneapolis, MN, USA). Medroxyprogesterone acetate (MPA) and Dulbecco’s modified Eagle’s medium: Ham’s F12 1:1 (DMEM) were purchased from Sigma Aldrich (Saint Louis, USA). RPMI 1640 was from Gibco (Thermo Fisher Scientific, CA, USA), trastuzumab was from Hoffmann-La Roche Ltd Genentech Inc. (Basel, Switzerland), and dimethyl sulfoxide (DMSO) was from Merck (USA). The antibodies are listed under Table S2.

Heregulin β1 (HRGβ1) and SDF-1 were purchased from R&D Systems, Inc. (Minneapolis, MN). Gefitinib and paclitaxel were from Selleck Chemicals, Inc. (Houston, TX). Antibodies used for western blot assays were from the following: HER3 (12,708), p-HER3 (Y1286), EGFR (4267), p-EGFR (Y1068), p-Akt (S473), Akt (9272), IGF-1R (3027), PARP (46D11), caspase-8 (1C12), caspase-3 (8G10) (Cell Signaling Technology, Inc., Danvers, MA); β-actin mouse mAb (clone AC-75) (Sigma-Aldrich, Inc., St. Louis, MO). Antibodies used for immunoprecipitation assays were from the following: HER3 (12,708), EGFR (4267), mouse IgG1 isotype control (5415), and rabbit IgG (2729) (Cell Signaling Technology, Inc.). Antibodies used for flow cytometry analysis were mouse anti-HER3 mAb (10,201-MM01) from Sino Biological, Inc. (Beijing, China). APC-labelled goat anti-mouse IgG Ab (405,308) from Biolegend Co. (San Diego, CA). All other reagents were from Sigma-Aldrich, Inc. unless otherwise specified.