Figure 4. Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells.
(a) N2a cells (3 × 105 cells) were cultured for two days in 25 cm2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10. Intensity of Bex1 (b), Bex2 (c), Bex3 (d), Bex4 (e) and Bex6 (f) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.