To create stable 32D cell lines expressing wild type human FLT3 and the nine FLT3/ITD variants, ecotropic retroviral transduction system was used (Clontech, Mountain View, CA, USA). The packaging cell line EcoPack 293 was transiently transfected with the retroviral pLXRN constructs using standard lipofectamine method (Lipofectamine 2000, Invitrogen, UK). 48 hours post transfection, the supernatants containing infectious retroviral particles were harvested and applied on the recipient 32D cells seeded at the cell density of 50,000/mL in RPMI 1640 with 10% FCS and supplemented with 1 ng/mL of mouse IL-3 (mIL-3; Sigma, Germany). Two days after retroviral transduction, 32D cell lines harboring ITDs were transferred into cultivation RPMI 1640 media supplemented with 10% FCS, without any mIL-3 added. 32D cell lines transduced with the pLXRN vector backbone and wt hFLT3 were cultivated in the presence of mIL-3, under G418 (Sigma, Germany) antibiotics selection. Polyclonal 32D populations – to exclude the effect of the retroviral integration site on the expression levels of transgenes – were subjected to further analyses.
Growth protocol
Large-scale cultivation of 32D cell lines for high-throughput transcriptome and proteome profiling Nine stable 32D cell lines carrying individual FLT3/ITD mutants, as well as parental 32D cell line, 32D cell line harboring wild type human FLT3 and 32D cell line carrying the pLXRN cloning vector were seeded into cultivation flasks at the cell density of 50,000/mL in RPMI 1640 cultivation media (Sigma, Germany) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, UK). The cultivation medium for the control 32D cell lines (parental, human wild type FLT3 and 32D harboring the cloning vector) were further supplemented with 1 ng/mL murine IL-3 (mIL-3, Sigma, Germany); FLT3/ITD-positive cell lines grew exponentially without mIL-3. 32D control cell line harboring wild type human FLT3 were split and cultivated in the presence or absence of FL (20 ng/ml, Sigma, Germany). Cells cultures were subjected to repeated passages at a constant cell density to achieve optimal growth rate. All cell lines were grown in the same lot of cultivation medium, serum, mIL-3, FL; in identical lot of cultivation flasks to make the cultivation conditions as uniform as possible. When the cultures reached sufficient density for both RNA and protein analyses (50 million for RNA analyses + 100 million for proteomics studies), individual 32D cell lines were repeatedly washed in 1xPBS (Sigma, Germany) to remove cultivation media and any traces of serum.
Extracted molecule
total RNA
Extraction protocol
50 million cells each were harvested into Trizol (Sigma, Germany). Trizol–isolated RNAs were DNA-ase I treated (NEB, UK) and re-purified using RNeasy MinElute Cleanup Kit (Quiagen, Germany). 100 million cells each from identical cultivation lots as those used for the RNA analyses were harvested and subjected to 5 gentle washes in 1x PBS to remove traces of sera from the cultivation media, the cells were lysed with the Pulverisette® 23 from FRITSCH GmbH (Idar-Oberstein, Germany). Subsequently, the cell debris was removed by centrifugation. The supernatant was acetone precipitated and solubilized in sample solution for 2-DE (9 M urea, 4% (w/v) CHAPS, 5 mM TBP, 1% Biolyte, 5% (v/v) protease inhibitor cocktail) at room temperature under gentle shaking for at least 1 h.
Label
biotin
Label protocol
1ug of total RNA were used to prepare biotinylated cDNA according to the standard GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual, including rRNA reduction step (Version 4, 701880 Rev. 4, Affymetrix).
Hybridization protocol
Fragmented and Labeled DNA Target was hybridized for 17 hr at 45C on GeneChip Mouse Exon 1.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 7G.
Description
Gene expression data from myeloblast-like cell line 32D transduced with wt FLT3
Data processing
The data were analyzed with Affymetrix Expression Console 1.1 and Bioconductor 2.1 and R statistics (version 2.6). Data were preprocessed using the RMA-Sketch normalization, probeset and metaprobeset summarization based on the default annotation from Affymetrix.
