Fig. 5: Mirin induces apoptosis through a p53-dependent mechanism in MNA cells.

a, b WB analysis of the indicated proteins and phosphoepitopes (a) and real-time PCR quantification of the indicated transcripts (b), in LAN5 cells, following mirin treatment for the indicated time points. Blots were probed with β-actin as a loading control. Transcripts expression was normalized on GAPDH levels and reported as fold induction compared to untreated controls. Data obtained by three independent experiments are reported as means ± SD. c, d MTS assay (c) and trypan blue exclusion test (d) performed in p53 mutant (SK-N-BE), p53 null (LAN1), and p53 wild-type (LAN5) MNA neuroblastoma cell lines, after mirin treatment. e LAN1 cells were transiently transfected with a p53-expressing or an empty plasmid. Apoptosis was evaluated by TUNEL assay (upper panel) and by measuring the amount of the cleaved form of PARP1 (c-PARP) via WB (bottom panel), after 15 and 5 h of mirin treatment, respectively. Average data obtained by three independent TUNEL assays are reported as fold induction compared to controls, ±SD. p was calculated by the ANOVA test. **p < 0.01. f LAN5 cells transiently expressing either p53 shRNAi (p53i) or control shRNAi were evaluated for cell death by the trypan blue exclusion assay (upper panel) and by measuring the amount c-PARP via WB (bottom panel) after 15 and 5 h of mirin treatment, respectively. Average data obtained by three independent cell death assays are reported as fold induction compared to controls, ±SD. p was calculated by the ANOVA test. *p < 0.05