RNA was purified from cells using the RNEasy Plus mini kit with a genomic DNA elimination step (Qiagen) and RNA converted to cDNA with the Quantitect Reverse Transcription kit (Qiagen) using instructions supplied by the manufacturer.
We analyzed the DNA contents by measuring the intensity of the fluorescence produced by propidium iodide using the FACSCalibur instrument (Beckon Dickinson, Mountain View, CA, USA) using instructions supplied by the manufacturer.
Briefly, after 24 h after incubation with 1.0 µM CG21680 or 1.0 µM ZM241385 or exposure to SMF, samples of medium (150 µl) were collected from cells and mixed with 130 µl dH2O and with 20 µl Griess reagent using instruction supplied by the manufacturer (Cat. No. G-7921, Invitrogen-Molecular Probes, Carlsbad, CA).
For reverse transcriptase reaction, the extracted RNA, random hexamers, and Superscript II reverse transcriptase (Invitrogen) were used according to the instructions supplied by the manufacturer.
Cells were transfected with 2 µg each DNA construct using Lipofectamine2000 (Invitrogen), according to instructions supplied by the manufacturer.
For quantification of PVX accumulation, leaf samples were homogenized as described by Murphy et al. [ 67] and used in double-antibody sandwich-ELISA [ 68] using reagents and antibodies and following instructions supplied by Bioreba (http://www.bioreba.ch/).
The steps in administering the Epipen® autoinjector were scored in the order of use according to the instructions supplied by the manufacturer: 1) recognizing the device; 2) removing the cap; 3) selecting the appropriate body site; 4) pressing the correct end of the device to the body; 5) pressing to activate; 6) holding in place for several seconds.
Transcriptional start sites of the GATA4 gene were mapped by rapid amplification of cDNA ends (5' RACE) using the GeneRacer kit according to instructions supplied by the manufacturer (Invitrogen).
Each of the seven W residues in the mature TPPI was altered to L residue according to the instructions supplied by the manufacturer using the primer pairs described in Table 2.
Total RNA from frozen tumour tissue samples were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the instructions supplied by the manufacturer.
After stabilization of the cells overnight, the cells were transfected with pooled siRNA for PDGFR-βor negative control siRNA (B-Bridge International, Sunnyvale, CA, USA) using LipofectAMIN 2000 (Invitrogen, Carlsbad, CA) according to the instructions supplied by the manufacturer.
