Bacterial artificial chromosome clones containing NAV3 DNA (RP11-36P3 and RP11-136F16; Research Genetics Inc., selleck bio Huntsville, AL, USA) and the chromosome 12 centromere probe (pA12H8; ATCC) were labelled with Alexa 594-5-dUTP and Alexa 488-5-dUTP (Invitrogen, Carlsbad, CA, USA), respectively, for tissue samples, or with digoxigenin and biotin, respectively, for cell lines. Detailed methods for probe labelling, slide preparation, and arm-specific MFISH are provided in Supplementary Online Materials, Method 2. The FISH results of the tissue samples were analysed by two blinded, independent researchers. Results are reported as the percentage of abnormal nuclei in 200 total nuclei as previously described (Ranki et al, 2011). For each cell line, 9�C47 metaphases were analysed for NAV3 and centromere 12, whereas 5�C11 metaphases were analysed for arm MFISH.

NAV3 LOH analysis using microsatellite markers and single-nucleotide primer ex tension NAV3 LOH assays were performed as previously described (Hahtola et al, 2008b). In addition, the A/G polymorphism (rs1852464) within exon 19 of the NAV3 gene, which exhibits up to 0.493 heterozygosity in Caucasians/Europeans, was used in the single-nucleotide primer extension reaction. Detailed methods are provided in Supplementary Online Materials, Method 3. Tumour samples were defined as showing LOH, if one allele had 40% more or less rs1852464 signal than matched normal tissue. In the event of constitutional homozygosity, LOH was assessed using flanking microsatellite markers (Cleton-Jansen et al, 2001).

Array CGH DNA was extracted from 50-��m paraffin-embedded tissue sections by standard protocols. Reference DNA was extracted from blood pooled at the Finnish Red Cross (Helsinki, Finland) from four healthy males and females after informed consent. DNA was then digested, labelled, and hybridised to a 244-K oligonucleotide array according to the manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA). Samples were scanned with a DNA microarray scanner and analysed using Feature Extraction v. 9.5.3.1 and CGH Analytics software v. 3.5.14 (Agilent Technologies). Analysis was performed using the Z-score and a 1-Mb moving average window. Log2-values under ��0.4 were not considered aberrant. Three colon carcinoma cell lines and two colon carcinoma tumour samples were analysed.

NAV3 gene silencing Batimastat in vitro The NAV3 gene was silenced with pooled siRNA oligonucleotides (On-Target SMART pool, Dharmacon, Chicago, IL, USA) as instructed by the producer. The CRL-1541 and CRL-1539 cell lines were transfected with 200pmol NAV3 pooled siRNA or scrambled control siRNA (Dharmacon), using Dharmafect1 transfection reagent (Dharmacon; for details see Supplementary Online Materials, Method 4). Efficient knockdown was confirmed by qPCR and microarrays (as shown in Supplementary Figure 1).