siRNA with equivalent %GC nucleotide content and FITC labelling was used as a control. Cells were assayed 24 h after siRNA duplex transfection. The effect of p65 suppression was monitored by p65 mRNA levels. RNA isolation and Real-Time PCR
Total RNA from cells subjected to different treatments was extracted using the RNeasy Mini Kit (Qiagen, Germany). RNA was quantified and the quality tested by photometric measurement on a Nanodrop apparatus (Wilmington, DE, USA). Only highly purified RNA (A260/A280>1.95) was used. cDNA synthesis was performed using the SuperScript™ III/RNaseOUT™ Enzyme Mix 2 and HSP990 50 μM oligo(dT) random primers (Invitrogen, Carlsbad, CA, USA). The cDNA was stored at −20°C. Oligonucleotide primers for the amplification were obtained from the Harvard Medical School Primer Bank ( http://pga.mgh.harvard.edu/primerbank/). The primer sequences used were as follows: p65 Forward Primer 5′-TTGAGGTGTATTTCACGGGACC-3′ and Reverse NU7026 chemical structure Primer 5′-GCACATCAGCTTGCGAAAAGG-3′, and GAPDH Forward Primer 5′-CCCATCACCATCTTCCAGG-3′ and Reverse Primer 5′-GAGATGATGACCCTTTTGGC-3′). PCRs were carried out in a final volume of 25 μl, containing 1 μM of both primers, 1x SYBR Green Supermix (Applied Biosystems), and variable amounts of cDNA templates. The program profile used for p65 amplification was the following: 95°C for 2 min, 45
cycles of denaturation for 30 sec at 95°C, annealing for 15 sec at 52°C and extension for 30 sec at 60°C. The program profile used for GAPDH was 95°C for 2 min followed by 45 cycles of denaturation, annealing and extension for 30 sec each at 95°C, 65°C and 60°C, respectively [26, 27]. Thermal cycling was performed in a Mx3000P™ real-time PCR system Stratagene Thermocycler (GE, USA). Data
were analysed with the accompanying software MX PRO System Software, using 2ΔΔCt EZH1/2 inhibitor formula. Statistical analysis Means and standard errors of the mean (SEM) were calculated. Significant differences between means were evaluated by analyses of variance and in the case of significance; a Newman–Keul’s post-hoc test was also applied. Real-time PCR data was analysed by a Student’s t-test. A difference was considered significant oxyclozanide when P was less than 0.05. SPSS+ version 13.0 statistical software was used. Results NAC and IFN-a decrease cell viability of liver cancer cells The ideal doses of IFN-α (2.5 x 104) and NAC (10 mM) were found through dose curves using concentrations ranging from 0 to 105 IU/mL for IFN-α, and 5 to 20 mM for NAC (data not shown). Both drugs had a dose-dependent effect. IFN-α at a concentration of 2.5 x 104 U/mL (96 hours) decreased cell viability to about 30% in HepG2 and Huh7 cells, while 10 mM NAC reduced cell viability in both cell lines at 48, 72, and 96 hours.