Lightcycler 2.0 apparatus

cDNA was synthesized using the AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA), 5 μl of hot-denatured DNA-free RNA, and 100 pmol of random hexamer primers. Products were precipitated in ethanol, resuspended in diethylpyrocarbonate-water, and stored at -20°C. Quantification was performed using a LightCycler 2.0 apparatus (Roche Diagnostics, Mannheim, Germany) and QuantiTect SYBR Green PCR Kits (Qiagen), using 2 μl of cDNA and 20 pmol of each primer. Primers (Additional file 1: Table S1) were synthesized by Eurofins MWG (Ebersberg, Germany).

To validate microarray data, we analysed SFN, FOXN1, Krt15, LHX2, CD34, and SOX9 gene expression by using mouse-predesigned oligos (Assay IDs: Mm.PT.58.42262048.g, Mm.PT.58.13135783, Mm.PT.58.5528981, Mm.PT.58.6480133, Mm.PT.58.8626728, and Mm.PT.58.42739087, resp., IDT®) and K6b-designed oligos (5′-CATCAAATACACCACCAGCG-3′ (forward) and 5′-AAGCAGCCAAAAAGAGAAGC-3′ (reverse)). The quantitative real-time PCR (RT-qPCR) was carried out using a LightCycler 2.0 apparatus (Roche®) and a DNA Master SYBR Green I kit (Roche). The templates were amplified in 45 cycles of a 3-step PCR process, which included 30 seconds of a denaturation step at 95°C, a 30-second primer-dependent annealing phase (60°C), and a 30-second template-dependent elongation at 72°C. The amplification of each template was conducted in duplicate in one PCR run. The differential expression of each mRNA was calculated as a ratio normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The data were analysed using the equation that was previously described by Livak and Schmittgen (amount of target = 2^−ΔΔCt [8 (link)]).

Total mRNAs were extracted from frozen tissues/cells using Nucleospin RNAII kit (Macherey‐Nagel), and 1 μg was reverse‐transcribed using the iScript cDNA Synthesis kit (BioRad). Real‐time quantitative RT–PCR was performed on a LightCycler 2.0 apparatus (Roche) using the Light Cycler FastStart DNA Master SYBERGreen I kit (Roche). Oligonucleotides sequences are available on request.
To assess impact of NF‐κB inhibition on netrin‐1 expression, OciLy3 cells were seeded into 6‐well plates at 3 × 10⁵ cells per well in a total volume of 2 ml of normal growth medium (see below). NFkB inhibitor (BAY 11‐7082) was diluted at a final concentration of 2 μM. Cells were harvested for RNA extraction after 6 hours of incubation with the inhibitor.