Apoptosis analyses

The Annexin V-binding assay (Immunostep, Salamanca, Spain) was used to measure the apoptosis induction following drug exposure. Cells were treated for 48 h with PLX4032, cDDP, TMZ, KP54 alone or with their simultaneous combinations. After washing with cold phosphate-buffered saline (PBS), cells were processed according to manufacturer’s protocol. Annexin V-binding was examined by flow cytometry (BD Accuri, Becton Dickinson, Milan, Italy) by acquiring ten thousand events for each sample. Instrument software (Becton Dickinson) was used to analyze the results.

Apoptosis was also evaluated by measuring the activation of caspase 3/7 as well as caspase 8 by luminescent Caspase Glo 3/7 assay System or Caspase-Glo 8 Assay System (Promega, Fitchburg). Cells were seeded in 96-well plates (7,000 cells/well in 100 µL of medium) and 24 h later treated with PLX4032, cDDP, TMZ or KP54 alone or with their simultaneous combinations. After 48 h, the activation of caspases was determined according to the manufacturer’s instructions. Relative luminescence units (RLU) were normalized with respect to the total protein content of each well to correct for the growth inhibitory effect of the treatment. Protein content was assayed by the BCA method.