c-Src control
Cells  MCF-10A cells (ATCC-CRL-10317) were obtained from the American Type Culture Collection and grown in complete growth medium [DMEM/F12 (Gibco) with 2 mM L-glutamine supplemented with 20 ng/mL epidermal growth factor (Sigma), 100 ng/mL cholera toxin (Sigma), 10 ng/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma) and 5% horse serum (Gibco)]. Cells were maintained at 37°C with 5% CO2.    Lentiviral constructs  The HA-tagged Src constructs are described elsewhere. Lentiviral particles for TetR-inducible expression of c-Src(wt) (LVP-Src-wt) and c-Src(mt) (LVP-Src-mt), as well as a short nonsense construct (LVP-mock) conferred resistance to Blasticidin S. LVP017-Neo allows constitutive expression of TetR and confers resistance to G418. Lentiviral particles were prepared by AMS Biotechnology (United Kingdom). Sequences were confirmed by capillary sequencing.    Generation of stably expressing cells  MCF-10A cells were grown to 50% confluence in complete growth medium. Cells were incubated with LVP017-Neo at a multiplicity of infection (MOI) of ten and lentiviral expression particles (MOI = 3) for 72h. Then, medium was replaced with fresh complete growth medium containing 60 g/mL G418 (Gibco) and 3.5 g/mL Blasticidin S (Invitrogen) for 14 days to select double-positive cells. Untransfected control cells were killed within 14 days when exposed to either 60 g/mL G418 or 3.5 g/mL Bsd. Double-positive cells were maintained in complete growth medium with 30 g/mL G418 and 1.75 g/mL Bsd. Expression of TetR mRNA was confirmed by qRT-PCR and was at comparable levels in the three engineered cell lines. The tetracycline-inducible system allowed regulation of transgene expression by adding tetracycline (Tet) to the cell culture medium. Inducible expression of the transgene in c-Src(wt)- and c-Src(mt)-transfected cells was verified by qRT-PCR and Western Blot. For the subsequent gene expression analyses we selected cell populations that showed similar levels of c-Src(wt) and c-Src(mt) expression at physiological levels.    Preparation of total RNA  Cells were grown to ~70% confluence in complete medium supplemented with 30 g/mL G418 and 1.75 g/mL Bsd. Tetracycline (Sigma-Aldrich) was added to the culture medium and cells were incubated for 24h. Then, total RNA was extracted with Trizol (Invitrogen) according to the manufacturer’s recommendations. The RNA was precipitated at -80°C and pelleted by centrifugation at 12,000 x g for 10 minutes. The pellets were washed with 75% ethanol, air-dried and solubilized in nuclease-free water. Remaining DNA was removed with the RNase-free DNase set from Qiagen according to the manufacturer’s protocol. Then, the solution was subjected to phenol-chloroform extraction. RNA integrity was assessed by agarose gel electrophoresis.     Primer design and synthesis  Primers for quantitative Real-Time PCR (qRT-PCR) were designed such that PCR products were smaller than 300 bp and intron-spanning, using the primer3 program (http://primer3.wi.mit.edu) and cDNA sequences retrieved from the UCSC Genome Browser and the current release of the human genome. The GAPDH primer pair has been described previously. The forward primer for mu SRC_HA is situated within the HA tag of c-Src constructs, c-Src(wt) and c-Src(mt), to avoid amplification of endogenous c-Src expressed by MCF-10A cells. Primers were synthesized by Metabion (Martinsried, Germany).
NEBNext Ultra RNA Library Prep Kit (New England Biolabs)