Figure 4

Roc-A-mediated chemoprotection depends on p53. (a) Roc-A inhibits Nutlin-3-induced p53 upregulation and protects T cells from Nutlin-3-induced cell death. T cells were treated with solvent (DMSO) or Nutlin-3 in the presence of Roc-A or solvent (DMSO) as indicated. After 24 h of treatment, the expression levels of p53 were analyzed by immunoblot (upper panel). Cell death was determined after 48 h of treatment by FSC/SSC profile (lower panel). Data are representative of three independent experiments. Error bars (S.D.) are shown. (b) Roc-A reduces Etoposide-induced mRNA expression of p53 target genes. T cells were treated with solvent (DMSO) or Etoposide (50 μM) in the presence of Roc-A (75 nM) or solvent (DMSO) for different time periods as indicated. Total RNA was isolated and subjected to quantitative real-time PCR for BAX, MDM2, BBC3 (PUMA), FAS and BCL2L11 (BIM). Data are presented as fold change in mRNA levels and normalized to control treatment with solvent (DMSO). Data are representative of three independent experiments. Error bars (minimum and maximum) are shown. (c) siRNA-mediated knockdown of p53 mimics the protective effect of Roc-A. T cells were transfected with scrambled (si-Ctrl.) or specific siRNA targeting p53 (si-p53). At 24 h after transfection, T cells were treated with solvent (DMSO) or Etoposide (50 μM) in the presence of Roc-A (75 nM) or solvent (DMSO) as indicated for 24 h. The expression levels of p53 were analyzed by immunoblot and cell death was determined by FSC/SSC profile. Data are representative of three independent experiments. Error bars (S.D.) are shown. (d) Roc-A-mediated protection is abolished in p53^−/− splenocytes. Splenocytes from p53^−/− or p53^+/+ mice were treated with 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for indicated time periods. Cell death was determined by DNA fragmentation. Data are an average of four independent experiments. Error bars (S.D.) are shown