Supplementary Material and methods
ERK and Akt status
For analyses of ERK and Akt status in cell cultures 31, 9, 7, 6, 13, 5, 38, 18, 35 and 21,
200,000 cells were seeded in 12 well plates (Sarstedt) in medium containing 10% FCS.
On the following day cells were incubated for 1 hour with our without 1 M imatinib. In
parallel, control samples were prepared from PAE cells expressing PDGF -receptor,
which were stimulated with or without 100 ng/ml PDGF-BB for 5 min. Cells were lysed
in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% deoxycholic acid, 0.5%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol, 200 µM Na3VO4.
Lysates were pre-cleared by centrifugation at 13,000 rpm for 15 minutes, heated for 5
min in SDS-sample buffer and subjected to SDS-PAGE using 12% polyacrylamide gels.
Proteins were transferred to Hybond-C-Extra membranes (Amersham Life Science) and
levels and phosphorylation of ERK and Akt was detected by immunoblotting with ERK
antibody (1 g/ml p44/42 MAP Kinase (Cell Signaling) )), Akt antibody (1 g/ml Akt
(Cell Signaling)), antibody recognizing phosphorylated ERK (1 g/ml Phospho-p44/42
MAPK (Thr202/Tyr204) (Cell Signaling)) and antibody recognizing phosphorylated Akt
(1 g/ml Phospho-Akt (Ser 473) (Cell Signaling)).
ERK and Akt expression and phosphorylation was quantified using the AIDA software,
version 3.10.039 (FUJIFILM). Experimental differences between membranes were
normalized by relating values from the glioma cultures with those from the control
samples present on each membrane. Expression values were also normalized for protein
content in the cell lysates, as determined by the BCA method (Pierce).
Amplification and labelling of RNA for micro-array analysis
RNA (5 g) from each cell line was used for linear amplification (Van Gelder et al.,
1990) and labelled with Cy-dye by in vitro transcription. Shortly cDNA was reversely
transcribed in a mixture of 5 g RNA, 1 l bacterial RNA cocktail, 1 l dT-T7 primer (1
g/ml, AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC
TTT TTT TTT TTT TTT), 4 l 5X Superscript II reaction buffer (Invitrogen), 2 l 1 M
dithiothreitol (Invitrogen), 1 l Ultrapure dNTP mix (Clontech), 1 l RNAsin (Ambion),
1 l template switch oligo primer (1 g/ml, AAA CAG TGG TAT CAA CGC AGA
GTA CGC GGG) and 2 l Superscript II (Invitrogen) at 42oC for 1 h. For second strand
synthesis, 106 l water, 15 l Advantage 10X PCR buffer (Clontech), 3 l of Ultrapure
dNTP mix, 1 l RNase H (Promega) and 3 l cDNA polymerase (Clontech) was added.
Samples were incubated at 37oC for 2 min to activate RNase H. Samples were incubated
at 94oC for 3 min to denature DNA, at 65oC for 3 min for primer annealing and at 75oC
for elongation by the cDNA polymerase for 30 min. Reaction was stopped by addition of
7.5 l of 1 M NaOH and 2 mM EDTA and incubation at 65oC for 10 min. The reaction
mixture was cleaned by phenol extraction, washed three times with water in a Microcon
YM-100 centrifugal filter (Millipore), and concentrated to a volume of 16 l. Anti-sense
RNA was generated by in vitro transcription with the MEGAscript T7 transcription kit
(Ambion). From the anti-sense RNA, cDNA was again reversely transcribed in a
Superscript II reaction, as described above, followed by generation of double stranded
DNA in a DNA polymerase reaction. In the second transcription reaction the UTP
nucleotide mix was substituted with a 1:2 mix of UTP and Cy-dye-UTP (Amersham
Biosciences). In parallel, a pool, composed of equal amounts of RNA from all the
cultures, was amplified to be used as reference in the subsequent hybridization
experiments.