Random primers

A minimum of five different RNA samples from GIST882 non-treated, treated 24h with 1 μM STI-571 and 48h 1μM STI-571 were used. Total RNA was extracted using RNeasy MiniKit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Genomic DNA was removed using the RNase- Free DNase set (Qiagen). RNA was reverse transcribed with 200 units of M-MLV Reverse Transcriptase (Invitrogen, Eugene, Oregon, USA) in a reaction containing 1μg of random primers (Amersham Bioscience, Piscataway, NJ, USA), 0.01M each dNTP, 1x First-Strand buffer and 0.1M dithiothreitol followed by heat deactivation. The cDNA reverse transcription product was amplified with specific primers (Supplementary Table 2) by qPCR using SYBR Green chemistry on a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Identical thermal profile conditions, namely 95°C for 10min, then 40 cycles of 95°C for 15sec and 60°C for 1min were used for all primer sets. Emitted fluorescence was measured during annealing/ extension phase and amplification plots were generated using the Sequence Detection System. Transcriptional quantification relative to GAPDH and β-actin reference genes was performed using qBase+ software (Biogazelle, Zwijnaarde, Belgium). Primers used are listed in Supplementary Table 3.

Total RNA was isolated from Arabidopsis cells using the RNeasy plant minikit (QUIAGEN S.p.A., Milan, Italy) according to the supplier’s recommendations. Residual DNA was removed from the RNA samples using a DNA-free kit (AMBION, Inc., Austin, TX, USA). Synthesis of cDNA was performed from 2 μg total RNA with 10 μM random primers (AMERSHAM Biosciences Europe GMBH, Milan, Italy), utilizing an Omniscript Reverse Transcriptase kit (QUIAGEN S.p.A., Milan, Italy) according to the supplier’s recommendations. PCR reactions were performed with specific primers for APX1 (X59600, 5′-GGACGATGCCACAAGGATAG-3′ and 5′-GGTTGCGATTTGAACACAT-3′); APX2 (X98275, 5′-ATTGCCGTTAGGCTTCTTGA-3′ and 5′-TACCAACCGACAAGGCTCTT-3′); APX6 (AV555486, 5′-CTGCTGGTGTGCTTCGTTTA-3′ and 5′-TTGAAAAACCATGGACGTCA-3′) and 18S rRNA (AJ236016, 5′-CATGATAACTCGACGGATCG-3′ and 5′-GAAGGCCAACGTAATAGGAC-3′). 18S rRNA was used as an internal control in order to normalize each sample for variations in the amount of initial RNA. For semi-quantitative RT-PCR, the cycle number in the linear range was empirically determined. These were analyzed on 1.5% agarose gel containing 0.5 μg mL^-1 ethidium bromide.