Etoposide

Sorafenib, etoposide, and APR-246 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), Calbiochem (VWR, Carnaxide, Portugal), and Sigma-Aldrich (Sintra, Portugal), respectively. All antibodies are listed in Table S5.

BO-1055 was synthesized at Dr. Tsann Long Su's laboratory at the Institute of Biomedical Sciences (IBMS), Academia Sinica, Taipei, Taiwan. PU-H71 was synthesized at Dr. Gabriela Chiosis' laboratory at Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, USA. Topotecan was purchased from Sigma-Aldrich (Saint Louis, MO), etoposide, melphalan and 4-hydroperoxy cyclophosphamide (4-HC) were purchased from Santa Cruz Biotechnology (Dallas, TX). SN38, Bendamustine hydrochloride, vincristine sulfate and cisplatin were purchased from Tocris (Bio-Techne, Minneapolis, MN). Doxorubicin and cyclophosphamide were purchased from the pharmacy at MSKCC.

The NSCs were seeded in 8-well chamber slides and coincubated with L. rhinocerus ME extract (5 or 50 μg/ml) and 100 μM DEX or 10 μM etoposide (Santa Cruz Biotechnology Inc.) for 48 h at 37°C in 5% CO₂. Cells cultured in medium without L. rhinocerus sclerotial extract, DEX and etoposide served as the negative control. etoposide was added as a positive control to induce apoptosis in NSCs. After treatment, NSCs were fixed with 4% (w/v) PFA for 15 min at room temperature and then incubated with PBS containing 0.1% (w/v) Triton X-100 for 15 min at room temperature. Next, 5 μg/ml Hoechst 33342 (Thermo Scientific Inc.) was added to the cells and incubated for 10 min at room temperature in the dark. The slide was then mounted and observed under a Nikon Eclipse Ti-S inverted fluorescence microscope. Images were captured using the Nikon NIS-Elements microscope imaging software.
Cells showing condensation and fragmentation of nuclei were considered apoptotic. For quantification of apoptotic nuclei, five random fields from each well were photographed and the number of total cells and cells with condensed or fragmented nuclei were determined by blind analysis. The percentage of apoptotic nuclei was calculated as the ratio of cells with condensed or fragmented nuclei to the total number of cells in each well.

Approximately 2000 compounds were screened from two different chemical libraries, including the Prestwick Chemical Library®, a collection of 1200 compounds at least in Phase II of clinical trials, and the BIOMOL’s FDA Approved Drug Library (Enzo Life Sciences), which consists of a collection of 640 FDA-approved drugs selected to maximize the chemical and pharmacological diversity. The compounds were supplied in 96-well plates as 10 mM (Prestwick®) and 2 mg/ml (BIOLMOL®) DMSO solutions. 5FU, DXR, daunorubicin quinacrine and methylene blue were purchased from Sigma-Aldrich and resuspended in DMSO. Epirubicin, idarubicin, pirarubicin, etoposide and teniposide were purchased from Santa Cruz, and valrubicin from Chemos.

Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration. Cells were treated with 25 uM Etoposide to induced Top2 DPCs and were detected essentially as described in Nitiss et al. (2012) (link), without modification.