Bosutinib

MCF-7 cells (ATCC), were cultivated as described in [3 (link)]. Primary mouse ear fibroblast were isolated and cultivated as follows:
Mouse ear notches or whole ears were collected and stored in cold Serum Free Medium (SFM), Advanced DMEM/F12 (Life Technologies) chopped with a sterile scalpel and incubated in SFM with 1mg/mL of collagenase A (Roche) for 1.5h at 37°C in 5% O₂. After digestion, the tissue was passed through an 18G syringe needle and then centrifuged for 10 minutes at 1000 rpm. Next, pellets were resuspended in Advanced DMEM/F12 medium with 10% FBS (SIGMA), 2mM L-glutamine (GIBCO) and a mix of 50U/mL penicillin and 10mg/mL streptomycin (GIBCO). Isolated cells were incubated and cultivated at 37°C in 3% O₂ and 5% CO₂.
Cells were treated with 100 or 200nM rapamycin (SIGMA) in serum containing medium for 3 days. Controls were treated with the same amount of DMSO. Bosutinib (Santa Cruz Biotechnology) was also prepared in DMSO and controls treated with DMSO only.

The SCREENWELL FDA‐approved drug library V2 containing 741 compounds was purchased from Enzo Life Sciences (Hayashi Kasei Co., Ltd.), and the International Drug Collection (IDC) containing 311 compounds was purchased from MicroSource Discovery Systems, Inc. (Namiki Shoji Co., Ltd.). Aldosterone (A9477), DHEA (D4000), dehydroisoandrosterone 3‐sulfate (D5297), and Reichstein's substance S (also called 11‐deoxycortisol; R0500) were obtained from Sigma. Androstenedione (A0845), cholesterol (C0318), corticosterone (C0388), estriol (E0218), hydrocortisone (H0533), and stanolone (also known as dihydrotestosterone; A0462) were purchased from Tokyo Chemical Industry (TCI). 17‐α hydroxypregnenolone (SC223186) and 21‐hydroxyprogesterone (also called deoxycorticosterone; SC231274) were obtained from Santa Cruz Biotechnology. Tyrosine kinase inhibitors imatinib mesylate (sc‐202180), nilotinib (sc‐202245), dasatinib (sc‐358114), bosutinib (sc‐202084), bafetinib (sc‐503249), and ponatinib (sc‐362710) were purchased from Santa Cruz Biotechnology, Inc. Chemical stocks (10 mM in DMSO or ethanol for cholesterol) were either obtained from the manufacturer (chemical libraries) or prepared (individual chemicals) and stored at −20°C.