Imatinib mesylate

Cardiac cells were prepared from ventricles of 1–3 dpp newborn pups and cultured as previously described.^{52 (link)}Freshly isolated newborn cardiac cells, pretreated or not for 30 min with 5  μM Imatinib-mesylate (sc-202180, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), were stimulated for 10 min with 100 ng/ml KL (R&D Systems, Inc., Minneapolis, MN, USA) before protein extraction.
Immunofluorescence on newborn neonatal myocytes was performed after 24 h of culture. Cells were fixed with 4% PFA, blocked with PBS/0.5% BSA/3% horse serum and stained overnight at 4 °C with mouse anti-sarcomeric myosin (1:5; MF20, Hybridoma Bank, Iowa, IA, USA); rabbit anti-MEF2C (1:100; Abcam: ab64644, Cambridge, UK) and goat anti-c-Kit (1:100; R&D) and then incubated 1 h at 37 °C with anti-rabbit FITC and anti-goat Cy5 secondary antibodies (1:300; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Images were acquired by Nikon Eclipse Ti - S microscope (Nikon Instruments S.p.A, Firenze, Italy).

K562 and KCL22 cells were obtained from Dr. Soumen Chakraborty lab (ILS, Bhubaneswar, India). The cells were cultured in 10% RPMI media with foetal calf serum (v/v) and 0.1 mg/ml PenStrep at 37 °C and 5% CO₂ conditions. Cells were passaged every 2–3 days and used for study till passage 20. The cells were tested for mycoplasma routinely and found negative consistently. Short-term TKI treatment was performed at inhibitory concentration 50 (IC₅₀) −0.75 μM of imatinib Mesylate (purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 48 h. The dead cells were washed and live cells were scored for Bcr-Abl activity by measuring the expression of p-Crkl, a downstream target molecule of Bcr-Abl tyrosine kinase (Supplementary Figures 1a and b). TKI-resistant K562 cell line was generated by gradual increase in the dose of TKI. The first treatment dose was 0.05 μM TKI, and gradually TKI dose was increased every week. The treatment was continued for 3 months till achievement of 5 μM and the cells were then characterized as TKI-resistant K562 cells. The TKI-resistant cells were then studied for further analysis.

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.