Real time-quantitative PCR (RT-qPCR)

RNA was extracted from the cells using a High Pure RNA Isolation Kit (Roche Applied Science, Castle Hill, NSW, Australia) and 1 μg of RNA was used in a 20 μL reverse transcription reaction (Transcriptor First Strand cDNA Synthesis Kit, Roche Applied Science, Castle Hill, NSW, Australia). RT-qPCR was performed in duplicate using SYBR Green I dye (Roche Applied Science, Castle Hill, NSW, Australia) with the LightCycler LC96 (Roche Applied Science, Penzberg, Germany). All primers were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Relative quantification using E-method established by Roche Applied Science was performed with β-actin mRNA as reference sequence and untreated samples as study calibrator. The cDNA samples were amplified using the following primer pairs with amplification efficiency (E): β-actin: 5′-ACTCTTCCAGCCTTCCTTC-3′(forward) and 5′-GATGTCCACGTCACACTTC-3′(reverse), E = 1.70; Mnk1: 5′-AAGGCCATTGAGACACTTCG-3′(forward) and 5′-CCCAAATGAAATAAAGCTCCTG-3′(reverse), E = 1.74; Mnk2: 5′-TCCTGCAGAGGTGGGACAGT-3′(forward) and 5′-ACGGTTCTGACCAGTCCTCC-3′ (reverse), E = 1.75.