Effects of IL-1beta gene silencing on CPT-induced senescence in U87-MG cells. A. Real- time analysis of IL-1beta gene silencing. IL-1beta mRNA levels were significantly reduced (** p < 0.01, Student's t-test) with respect to mRNA level of the non silenced CPT-treated cells. IL1-B relative expression was obtained by performing the comparative method . Firstly, data were normalized to GAPDH, then to a calibrator, consisting of mRNA obtained from CPT treated cells. The results were expressed as 2^-ΔΔCt, where ΔCt of each sample was defined as Ct(target gene = IL1-beta) - CtGAPDH, and ΔΔCt = ΔCt(sample = cDNA from CPT+iRNAs treated cells) - ΔCt calibrator(cDNA from CPT treated cells). ΔΔCt calibrator is always equal to 0 so that 2^-ΔΔCt is always 1. B. SA-β gal activity, in response to 1 μM CPT, was quantified in U87-MG cells in the presence or in the absence of IL-1beta gene silencing. DMSO, solvent control. Bars, standard error. * p < 0.05, Student's t-test. C. 250 cells from either CPT and solvent exposed cells or IL-1beta-silenced/CPT-treated U87-MG cells were sub-cultured in fresh medium in five replicates. Cells were maintained in culture for 10 days, with bi-weekly medium changes, then were fixed with methanol, stained with 10% aqueous Giemsa and scored for colony formation. Only colonies containing more than 50 cells were counted. Clonal efficiency was calculated as the mean number of colonies per plate with respect to the solvent. Bars, standard error. * p < 0.05, Student's t-test. ** p < 0.01, Student's t-test.