Each subgroup contained 6 repeated
pores. The transduction efficiency was quantified using fluorescence microscopy and flow cytometry 48 Selleck Caspase inhibitor h after transduction. Detection of HA117 and MDR1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) 48 h after transduction, total RNA was extracted from cells in the experiment and control groups using the Tripure isolation reagent (TianGen Biotechnology, China) according to the manufacturer’s instructions. RT was performed using the TaKaRa RT kit (TaKaRa Biotechnology, China). The expression HDAC inhibitors in clinical trials levels of mRNA were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). The relative expression levels of the target genes were determined by calculating the Wnt pathway fluorescence intensity ratio between the target gene and GAPDH. The primers used for PCR were designed according to the information from the human genomic data base and were synthesized by Invitrogen Biotechnology Company (USA). The sequences of the primers used for the amplification of HA117, MDR1 and GAPDH were as follows: HA117- (forward) 5′-CAGAGTCAGGGACTTCAGCCTTAT-3′, (reverse) 5′-CTGTTTCCTTCTCACTCCCAACCA-3′; MDR1- (forward) 5′-GCTGGTTTGA TGTGCACGATGTTGG-3′, (reverse) 5′-ATTTTGTCACCAATTCCTTCATTAA-3′; GAPDH- (forward) 5′-ACCACAGTCCATGCCATCACT-3′, (reverse) 5′-TCCACCACCCTGTTGCTG TA-3. The PCR conditions were as follows: denaturation at 94°C for
5 min and 33 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, and a final extension at 72°C for 5 minutes. The PCR products were resolved on 2% agarose gels, and the gels were photographed. Densitometric analysis was performed using the UVP gel image analysis system (Bio-Rad, USA). Detection of P-gp expression using a western blotting The cells were harvested and lysed in lysis buffer (0.5% Nonidet P-40, 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1 mM Na3VO4) supplemented with protease inhibitors and 1 mM phenylmethylsulfonyl fluoride (PMSF). Approximately 100 μg of total cellular lysate was then subjected to standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For western blotting analysis,
the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Inc., USA), which were then blocked for 1 h with 8% non-fat milk in 10 mM Tris-HCl pH 7.5, 100 mM NaCl, and Phosphoglycerate kinase 0.1% (w/v) Tween 20. The membranes were first incubated with antibodies against β-actin or P-gp (both from Santa Cruz, USA) overnight at 4°C, followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibody. The protein signals were detected using an enhanced chemiluminescence kit (BiYunTian Biotechnology, China) and analyzed using the Bio-Rad (USA) imaging system and associated software according to the manufacturer’s instructions. Drug elimination experiments The activities of HA117 and MDR1 were determined using the daunorubicin (DNR) efflux assay.