Quantstudio design analysis software v1

Total RNA was extracted from hTERT-RPE cells treated with DMSO or BFA using RNeasy^R mini kit (Qiagen). DMSO or BFA was treated 30 min before serum-restimulation at the indicated concentrations. cDNA was generated by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Conventional RT-PCR was performed using a ProFlex™ Base Thermal Cycler (Applied Bio-systems) with the conditions of 95°C for 20 s, 62°C for 30 s, and 72°C for 45 s for a total of 25 cycles for Plk1, Dvl2, and GAPDH, followed by a 10 min final extension at 72°C. PCR products were electrophoresed in 3% agarose gel, stained with EcoDye™ Nucleic Acid Staining Solution (BIOFACT, Korea), and photographed. Real-time RT-PCR was performed in a final volume of 20 μl with 2 μl of cDNA, 10 pmol forward and 10 pmol reverse primer in 1X power SYBG green PCR master Mix (Applied Biosystems, USA) with the condition of 95°C for 15 s for denaturation, 55°C for 1 min for annealing and 72°C for 15 s extension using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The expression value of each gene was normalized by that of GAPDH. Final values were calculated using the ΔΔCt method. The results were analyzed using QuantStudio™ design & Analysis software v1.4 (Applied Biosystems). All the primers used in these experiments are summarized in Supplementary Table S3.