Etoposide

Doxorubicin hydrochloride (#D1515), vincristine, and insulin growth factor 2 (IGF2, #I2526) were purchased from Merck. Irinotecan (#S2217), MK-2206, capivasertib (AZD5363), alpelisib (HY-15244), nilotinib (S1033), pazopanib (S3012), and ponatinib (S1490) were purchased from Selleckchem. Everolimus (RAD001, SRP020750e) was purchased from Sequoia Research Products, etoposide was purchased from Sandoz and AVE 1642 from Immunogen. D-188514, an ifosfamide analog that does not require metabolic activation, was purchased from Niomech. The PI3K/mTOR dual inhibitor NVP-BEZ235 was kindly provided by Novartis. Trabectedin (ET-743) was kindly provided by PharmaMar. Working dilutions of all drugs were prepared immediately before use.

The A375M and WM793B cells were treated with various doses of γ-irradiation (from 5 to 30 Gy, at the average dose rate 2 Gy/min) using ⁶⁰Co source at the Ruđer Bošković Institute in Zagreb, Croatia or with cisplatin (Sigma-Aldrich/Merck) or etoposide (Sandoz, Holzkirchen, Germany) (both IC50 of 1 μM, defined with MTT assay). Alternatively, doxorubicin (0.75–1.5 μM, Sigma-Aldrich/Merck) and dacarbazine (5-10 μM, Sigma-Aldrich/Merck) were used to treat most of the cell lines. Cells were harvested for protein analysis at different time points and protein extracts were analyzed by Western blotting. In addition, A375M and WM793B were treated for 24 h with different doses of vemurafenib (1 µM, 2 µM, or 5 µM) where DMSO treatment was used as a negative control.

Cells were cultured with titrated concentrations of ABT-199 (Cat#A-1231, Active Biochem), A1331852 (Lessene Lab, WEHI), S63845 (Cat#A-6044, Active Biochem), MDM2 inhibitor RG7388 (Cat#C-1287, Chemgood), cisplatin (Sandoz), or etoposide (Sandoz) for 24–48 h. They were then suspended in KDS-BSS buffer containing 2 µg/ml propidium iodide. Cell viability was measured by assessing the exclusion of propidium iodide staining using a flow cytometer (FACS Calibur; BD Biosciences).