5×105 cells/well. Total RNA was extracted from CCA cell lines using

TRIzol® reagent following the manufacturer’s instructions (Invitrogen). Total RNA was isolated using a previously Verteporfin described method [20]. Total RNA (1 μg) was reverse transcribed in a 20 μL reaction mixture, containing 0.5 μg of oligo(dT)15 primer, 20 U of RNasin® ribonuclease inhibitor, and 200 U of ImProm-II™ reverse transcriptase in BIBF 1120 price 1× PCR buffer, 3 mmol/L MgCl2, and 1 mmol/L dNTPs. The first-strand cDNA was synthesized at conditions of 42°C for 60 min. The reverse transcription products served as templates for real-time PCR. PCR amplification was performed using specific primers for the NQO1, wild type p53 and the internal control using β-actin. The primer sequences were as follows: 1) NQO1 (NM_000903.2): forward primer 5’-GGCAGAAGAGCACTGATCGTA-3’ and reverse primer 5’-TGATGGGATTGAAGTTCATGGC-3’;

2) wild type p53 (NM_005256778.1) [25]: forward primer 5’-ATGGAGGAGCCGCAGTCAGATCC-3’ and reverse primer 5’-TTCTGTCTTCCCGGACTGAGTCTGACT-3’; 3) β-actin: forward primer 5’-TGCCATCCTAAAAGCCAC-3’ and reverse primer 5’-TCAACTGGTCTCAAGTCAGTG-3’. The real-time fluorescence PCR, based on EvaGreen® dye, was carried out in a final volume of 20 μL containing 1x SsoFast™ EvaGreen® supermix (#172-5200; Bio-Rad, CA, USA), 0.5 μmol/L see more of each NQO1 or wild type p53, and 0.25 μmol/L of β-actin primer. Thermal cycling was performed for each gene in duplicate on cDNA samples in 96-well reaction plates using the ABI 7500 Sequence Detection system (Applied Biosystems). Nintedanib A negative control was also included in the experimental

runs. The negative control was set up by substituting the template with DI water. Real-time PCR was conducted with the following cycling conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 31 s. To verify the purity of the products, a melting curve analysis was performed after each run. Upon completion of 40 PCR amplification cycles, there was a dissociation step of ramping temperature from 60°C to 95°C steadily for 20 min, while the fluorescence signal was continually monitored for melting curve analysis. The concentration of PCR product was calculated on the basis of established standard curve derived from serial dilutions of the positive control for NQO1, wild type p53 and β-actin in the CCA cell lines. Western blot analysis After treatment with chemotherapeutic agents, CCA cells were washed with PBS, collected, and lysed at 4°C with 1x cell lysis buffer with 1 mmol/L dithiothreitol and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) with vigorous shaking. After centrifugation at 12,000 g for 30 min, supernatant was collected and stored at -80°C until use. Thirty microgram of the protein samples were mixed with 5x loading-dye buffer, heated at 90°C for 10 min, and proteins were then separated by electrophoresis in 10% SDS-polyacrylamide gel.