Rt premix kit

Total cellular RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed with 1 μg RNA using RT-Premix kit (ELPIS Biotech, Daejeon, South Korea) according to the manufacturer’s instructions. The primers used for SYBR Green real-time quantitative (q) PCR were as follows: E-cadherin forward 5′-TACACTGCCCAGGAGCCAGA-3′ and reverse 5′-GGCACCAGTGTCCGGATTA-3′; Snail, forward 5′- GACCACTATGCCGCGCTCTT-3′ and reverse 5′-TCGCTGTAGTTAGGCTTCCGATT-3′; GAPDH, forward 5′- GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′ [24 (link)]. The reaction was performed using an Applied Biosystems^® 7500 96-Well Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). After normalization to the GAPDH gene, the expression levels for each target gene were calculated using the comparative threshold cycle method. The data are presented as the mean ± standard deviation (SD) from three independent experiments.

IWR-1 was obtained from ENZO (Farmingdale, NY, USA), PI3K inhibitor LY294002 was purchased from Sigma-Aldrich (St Louis, MO, USA), and recombinant human tumor necrosis factor (TNF)-α was from R&D Systems (Minneapolis, MN, USA). Primary antibodies against E-cadherin, N-cadherin, Snail, Survivin, Vimentin, p-Akt (Ser473), t-Akt, β-catenin, and β-Actin as well as secondary antibodies conjugated to horseradish peroxidase (HRP), Alexa-594, and FITC were obtained from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 2000, SYBR Green, and the total RNA purification kit were purchased from Invitrogen (Carlsbad, CA, USA), and the RT-Premix Kit was obtained from ELPIS Biotech (Daejeon, South Korea).