Topoisomerase 1 relaxation assay kit

The APOPercentage™ kit was acquired from Biocolor Ltd. (Carrickfergus, Ireland); the Caspase-Glo^®-3/7 assay kit, MitoToxGlo^™, and MultiTox-Glo multiplex kit were acquired from Promega (Madison, WI, USA); the Topoisomerase-I relaxation assay kit was acquired from TopoGen (Buena Vista, CO, USA); the Annexin V-FITC apoptosis kit was acquired from BD Pharmingen (San Diego, CA, USA); the 5- (and 6) -chloromethyl-2′,7′-dichlorofluorescein diacetate acetyl ester (CM-H₂DCFDA) and MitoTracker Red (CMXRos) were acquired from Molecular Probes (Invitrogen, OR, USA); the DAPI (4′,6-diamidino-2-phenylindole), TRITC-conjugated phalloidin, and anti-vinculin were acquired from Merck Millipore (Darmstadt, Germany); Hank’s balanced salt solution (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM), and phosphate buffered saline (PBS) were acquired from Gibco (USA); penicillin/streptomycin, Triton X-100, Tween-20, and paraformaldehyde were acquired from Sigma-Aldrich (St. Louis, MO, USA); and NucBlue Live ReadyProbes^TM Reagent was purchased from Thermo Fischer (Johannesburg, South Africa). All other chemicals and reagents used in this study were of analytical grade.

The cell membrane integrity and cellular ATP levels were assessed following the manufacturer’s instructions for the Topoisomerase-I relaxation assay kit (TopoGen, Buena Vista, CO, USA). The four cell lines were plated in a 96-well white walled plate (100 µL/well) at 5 × 10⁴ cells/mL in galactose-fortified glucose and FBS-free medium. The cells were treated with increasing concentrations (0–10 µg/mL) of the compounds for 2 h. The cells were first incubated with bis-AAF-R110 substrate for 30 min to measure the mitochondrial toxicity, and the fluorescence signal was recorded at 485 nm excitation and 585 nm emission wavelengths. Then, ATP detection reagent was added and further incubated for 5 min, after which the luminescence signal was measured.