The signal intensity of each find more band was measured and expressed in optical density (OD). The semi-quantitative analysis of telomerase activity was performed by adding the signals of the ladder products in each lane, corrected for the background. RT-PCR The expression of hTERT mRNA was semi-quantitatively evaluated by RT-PCR amplification as described [20]. Briefly, hTERT mRNA was amplified using the primer pairs: 5’-CGGAAGAGTGTCTGGAGCAA-3’

and 5’- GGATGAAGCGGAGTCTGGA-3’ . Total RNA was isolated from the cells using Trizol (Invitrogen) Selleckchem LY3039478 according to the manufacturer’s protocol, and cDNA was synthesized from 1 μg of RNA using the cDNA Cycle kit (Invitrogen) with random primers. Typically, 2 μl aliquots of the reverse-transcribed cDNA were amplified by 28 cycles of PCR in 50 μl of buffer [10 mM Tris–HCl (pH 8.3), 2.5 mM MgCl2, and 50 mM KCl] containing 1 mM each of dATP, dGTP, dTTP, and 32P- dCTP (Amersham Biosciences, Amersham, United VX-689 ic50 Kingdom), 2.5 units of Taq DNA polymerase (Promega, Madison, Wisconsin), and 0.2 mM primers. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 45 s. The PCR products were resolved by electrophoresis in 7% polyacrylamide gels. The efficiency of cDNA synthesis from each sample was estimated by PCR using

GAPDH specific primers: 5’ – GAAGGTGAAGGTCGGAGTC-3’and 5’-GAAGATGGTGATGGGATTTC-3’. Real time PCR Briefly 2 × 106 viable Jurkat T cells treated or not with saquinavir were harvested after 24 h incubation. Samples were resuspended in 1 ml Trizol (Ambion)

and RNA samples were extracted according to the manufacturer’s instructions. Two μg of RNA were purified by clearance of DNA traces using Turbo DNA-free kit (Applied Biosystems, Life Technologies, Monza, Italy). cDNA was synthesized using 2 μg of DNA-free RNA and TaqMan RT kit (Applied Biosystems), according to the manufacturer’s instructions. hTERT mRNA was quantitatively detected by real-time reverse transcription polymerase chain reaction (RT-PCR). For quantitative real time RT-PCR 5 μl (i.e. 2 μg) of cDNA/sample was amplified according to the manufacturer’s instructions (Applied Biosystems) on a Real-Time Stratagene MX3005P, using a TaqMan gene expression assay kit (Applied Biosystems, code Endonuclease # Hs00162669-m1). Levels of hTERT were normalized against GAPDH housekeeping expression (Applied Biosystems code # 4326317E). All real-time RT-PCR reactions were performed in triplicate. Normalized TERT expression (TERT/GAPDH) was calculated using the ΔΔCt method according to the supplier’s protocol. Electophoretic mobility shift assay (EMSA) The binding of the transcription factor c-Myc to its specific downstream E-Box DNA binding-site from hTERT promoter was analyzed by EMSA [21]. In particular we analyzed the DNA oligonucleotide 5’- TCCTGCTGCGCACGTGGGAAGCCCT-3’, containing the downstream “CACGTG” E-Box sequence localized at position −34 of hTERT promoter.