Fig. 3

Quarfloxin and CX-5461 induce p53-dependent apoptosis in TP53-wt neuroblastoma cells. a Representative western blot (WB) for the assessment of cleaved PARP (c-PARP) and p53 expression in neuroblastoma cell lines treated for 24 h with 150 nM quarfloxin (left panel) or 230 nM CX-5461 (right panel) or 0.1% vehicle (DMSO and DMF, respectively). b Representative WB evaluation of cleaved-Caspase-3 (c-Casp-3), p53, and p21 expression in neuroblastoma cell lines treated as in a. c FACS analysis of Annexin V-positive neuroblastoma cells after 24 h treatment with 150 nM quarfloxin or 230 nM CX-5461 or vehicle. The data represents the mean and SD of representative experiments performed with two biological replicates pr. condition (ns, not significant; **p ≤ 0.01; ***p ≤ 0.001). d p53 induction sensitizes neuroblastoma cells to quarfloxin and CX-5461. The day after transfection with siRNAs targeting TP53 (siTP53-1 and siTP53-2), 50 nM quarfloxin (left panel) or 75 nM CX-5461 (right panel) were added and cell viability was measured 48 h after treatment. The viability of vehicle + respective siRNA was set to 100%, and quarfloxin and CX-5461 treated cells were normalized against their respective controls. The data represents the mean cell viability and s.d. of two individual experiments performed in duplicate (***p ≤ 0.001; ****p ≤ 0.0001). e Representative WB assessing c-PARP levels after p53 knockdown. IMR-32 cells were reverse transfected with siTP53-1 and siTP53-2, and treated with 150 nM quarfloxin (left panel) or 230 nM CX-5461 (right panel) for 24 h before harvesting and immunoblotting. f Assessment of p53 transcriptional activity. Neuroblastoma cells were co-transfected immediately after seeding with a p53-responsive firefly luciferase reporter (pg13) and CMV-renilla luciferase. 150 nM quarfloxin or 230 nM CX-5461 were added the following day and cells were treated for 24 h before harvesting and analysis. Pg-13 expression was normalized to Renilla expression (RLU) and the data shown represents the mean of one experiment with three biological replicates and is representative of two independent experiments (ns, not significant; **p ≤ 0.01; ***p ≤ 0.001)