Sch772984

Heregulin β-1 (Sigma, H7660) was used at a working concentration of 20 ng.mL⁻¹. For lysosome and proteasome inhibition, cells were treated for 4 h with 50 nM of bafilomycin A1 (Calbiochem, 196000) and 1 µM of bortezomib (Adooq Bioscience, A10160), respectively. CHX (Sigma, 01810) was used for translation inhibition at 25 µg.mL⁻¹. For targeted inhibition of intracellular signaling proteins, trametinib (Adooq Bioscience, JTP-74057, [100] nM), rapamycin (Santa Cruz Biotechnology, sc-3504A, [100] nM), buparlisib (Adooq Bioscience, AT11016, [500 nM]), selumetinib (Adooq Bioscience, A10257, [1 µM]), SCH772984 (Adooq Bioscience, A12824, [1] µM) and MK-2206 (Adooq Bioscience, A10003, [2] µM) were used for the indicated time points.

The following reagents were used: RNase (Carl Roth, Karlsruhe, Germany); PhosSTOP™ EASYpacks phosphatase inhibitors cocktail and cOmplete™ EASYpacks protease inhibitors cocktail (Roche, Mannheim, Germany); CM-H₂DCFDA (Molecular Probes, Invitrogen, Eugene, OR, USA); N-acetyl cysteine and GSK-690693 (Akt inhibitor) (MedChemExpress, Monmouth Junction, South Brunswick, NJ, USA); Anisomycin (NeoCorp, Weilheim, Germany); carboplatin, cisplatin, and docetaxel (Pharmacy of the University Hospital Hamburg-Eppendorf, Hamburg, Germany); propidium iodide (PI), CCCP (2-[2-(3-chlorophenyl)hydrazinylyidene]propanedinitrile), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, Taufkirchen, Germany); enzalutamide (Hycultec GmbH, Beutelsbach, Germany); PD98059 (MEK inhibitor) (Merck Chemicals GmbH, Darmstadt, Germany); SCH772984 (ERK1/2 inhibitors) (Adooq Bioscience, Irvine, CA, USA); olaparib (PARP inhibitor), SP600125 (JNK1/2 inhibitor), and SB203580 (p38 inhibitor) (LC Laboratories, Woburn, MA, USA); annexin-V-FITC (BD Bioscience, San Jose, CA, USA). The primary and secondary antibodies used are listed in Table 3.

For signaling pathway inhibition experiments, cells were treated with vehicle (0.1% DMSO) or PI3K inhibitor LY294002 (50 μm) (Promega, Madison, WI, USA), p38 MAPK inhibitor SB203580 (10 μm) (Promega), MEK inhibitor U0126 (10 μm) (Promega), PD98059 (20 μm) (Promega), Raf‐1 kinase inhibitor ZM‐336372 (1‐5 μm) (Enzo Life Sciences, Farmingdale, NY, USA), or ERK inhibitor SCH772984 (1 μm) (AdooQ BioScience, Irvine, CA, USA) for 48 h. For DNA demethylation experiments, cells were treated with vehicle (0.1% ethanol) or 5 μm 5‐aza‐2′‐deoxycytidine (Sigma) for 48 h.