MDCK-II cell lines were obtained from the American Type Culture Collection (LGC Standards-SLU, Barcelona, Spain) and grown in DMEM media (Gibco BRL), supplemented with 10% fetal bovine serum, 10 mmol/L glutamine (Life Technologies), 100 µg/mL ampicillin and 32 µg/mL gentamicin (or 100units/ml penicillin/streptomycin for MDA-MB-231 cells). All cell lines were grown at 37°C in a humidified 5% CO2 atmosphere. Stable transfectants were obtained from parental MDCK-II after transfection with pcDNA3-hLOXL2-Flag, pcDNA3-ΔLOXL2-Flag, Cells were grown in the presence of G418 (400 μg/ml) for 3-4 weeks and individual clones isolated with cloning rings. At least 10 independent clones were isolated from each transfection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was using Trizol (Life Technologies, Barcelona, Spain) following the manufacturer’s protocol and subsequently treated with DNase (Promega, Barcelona, Spain). Total RNA concentration and purity was assessed using Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and subsequently retro-transcribed using random hexamer primers and cDNA First Strand Synthesis kit (MRI Fermentas, Hanover, MD, USA)
Label
Cy5
Label protocol
Four independent passages from stably-transfected of each analysed tranfectec cells (LOXL2, DLOXL2) and empty-pCDNA3.1 vector, used as control. Total RNA (1mg)from cell lines was isolated using Trizol reagent (Life Technologies) as indicated by the manufacturer. Purity of isolated RNA was evaluated spectrophotometrically by the A260/A280 absorbance ratio. RNA was labeled and array hybridized using the Low RNA Linear Amplification Kit and the In Situ Hybridization Kit Plus (Agilent technologies), respectively, following manufacturer’s protocol.
reference: Pool from MDCK pcDNA3 control cells tissue: Ephitelial
Growth protocol
MDCK-II cell lines were obtained from the American Type Culture Collection (LGC Standards-SLU, Barcelona, Spain) and grown in DMEM media (Gibco BRL), supplemented with 10% fetal bovine serum, 10 mmol/L glutamine (Life Technologies), 100 µg/mL ampicillin and 32 µg/mL gentamicin (or 100units/ml penicillin/streptomycin for MDA-MB-231 cells). All cell lines were grown at 37°C in a humidified 5% CO2 atmosphere. Stable transfectants were obtained from parental MDCK-II after transfection with pcDNA3-hLOXL2-Flag, pcDNA3-ΔLOXL2-Flag, Cells were grown in the presence of G418 (400 μg/ml) for 3-4 weeks and individual clones isolated with cloning rings. At least 10 independent clones were isolated from each transfection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was using Trizol (Life Technologies, Barcelona, Spain) following the manufacturer’s protocol and subsequently treated with DNase (Promega, Barcelona, Spain). Total RNA concentration and purity was assessed using Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and subsequently retro-transcribed using random hexamer primers and cDNA First Strand Synthesis kit (MRI Fermentas, Hanover, MD, USA)
Label
Cy3
Label protocol
Four independent passages from stably-transfected of each analysed tranfectec cells (LOXL2, DLOXL2) and empty-pCDNA3.1 vector, used as control. Total RNA (1mg)from cell lines was isolated using Trizol reagent (Life Technologies) as indicated by the manufacturer. Purity of isolated RNA was evaluated spectrophotometrically by the A260/A280 absorbance ratio. RNA was labeled and array hybridized using the Low RNA Linear Amplification Kit and the In Situ Hybridization Kit Plus (Agilent technologies), respectively, following manufacturer’s protocol.
Hybridization protocol
In Situ Hybridization Kit Plus (Agilent technologies) protocols
Scan protocol
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version 10.0).
Data processing
Agilent Feature Extraction Software (v 10.0) was used for background subtraction and LOWESS normalization.
