Cao et al. BMC Cancer 2010, 10:68 http://www.biomedcentral.com/1471-2407/10/68 Table 1 Northern blot probe primers Gene symbol ARMER PCBP1 PDHB FUBI b-actin F-primer R-primer F-primer R-primer F-primer R-primer F-primer R-primer F-primer R-primer Primer sequence CGGAGGGAGATAATCGCAGCACCAAC GAAGCAGGTTGTGGACTTGTTGTCCC CGCCGGAATTGACTCCAGCTCTCC CAGAAAGGGGTTATTGAGGGAACCTAC CGGTGTCTGGCTTGGTGCGGAGAC GAGTTTATAACCTGGTCAATGGCTTGC TTTCAGATTCTAGAGTTATGTCCTC ATAGAATGGGATGCTGTCTGGGAA CGCGCTCGTCGTCGACAACGGC CGTACATGGCTGGGGTGTTGAAGGTC M NaCl and 0.5 M Tris-HCl (pH 8.0), 0.2 M Tris-HCl (pH 7.5) and 2 × SSC buffer, respectively. Subtracted and unsubtracted PCR products were used as probes labeling with digoxigenin-dUTP at 37°C overnight according to the DIG High Prime Labeling Kit protocol (Roche Molecular Biochemicals, Mannheim, Germany). The dot blot hybridization was performed overnight at 42°C using the DIG Easy Hyb system (Roche Molecular Biochemicals, Mannheim, Germany) following the man- ufacturer’s instructions. The blots were washed at RT twice at low stringency (2 × SSC, 0.1% SDS) for 5 min and then at 68°C twice at high stringency (0.1 × SSC, 0.1% SDS) for 15 min. Hybridized probes were subse- quently detected by alkaline phosphatase-conjugated anti-DIG antibody and the CSPD chemiluminescent detection system (Roche Molecular Biochemicals, Man- nheim, Germany). Sequencing and Sequence analysis Nucleotide sequences were compared with the GenBank using the BLASTn programs from the National Center for Biotechnology Information http://www.ncbi.nlm.nih. gov/BLAST/ and the BLASTx for the protein. Protein patterns were then identified by searching the PROSITE database http://www.expasy.ch/tools/ and Pfam database http://pfam.sanger.ac.uk/. The DNA was sequenced from both strands on an ABI PRISM 377 Genetic Analyzer (PE Biosystems, Fos- ter City, CA, USA). Northern blotting analysis Digoxigenin-labelled DNA probes were applied for Northern blotting analysis. Oligonucleotide primers for the DNA probes were designed based on the known sequences (Table 1). The programs were as follows: 94° C for 5 min, followed by 35 cycles of 30 sec at 94°C, 30 sec at 56°C, and 90 sec at 72°C, and a final polymeriza- tion step at 72°C for 10 min, and then the PCR products were purified using QIAquick PCR Purification Kit Page 4 of 11 (Qiagen, Hilden, Germany). The fragments were labeled with digoxigenin-dUTP as above. Each set of total RNA was extracted from conditioned culture non-SP single clone cells and non-conditioned culture single clone cells described above. Total RNA was quantified on a spectrophotometer at 260 nm. Ali- quots containing 10 μg of total RNA corresponding to each tissue were mixed with glyoxal and DMSO and heated at 50°C for 30 min and then placed on ice over 1 min. The treated RNAs were then separated through a 1% agarose (Seakem) gel. The intensity of ethidium bro- mide-stained rRNA bands was examined visually to ensure that equal amounts of total RNA were loaded onto the gels. The RNA was then transferred to a nylon membrane (Hybond-N, Amersham Pharamacia Biotech, Uppsala, Sweden), and UV cross-linked. Hybridization was performed overnight at 50°C, washed at RT twice at low stringency for 5 min and then at 55°C twice at high stringency for 15 min and were detected as above. Results Identification of SP cells in the CNE-2 single clones To determine whether different percentages of SP cells were contained in CNE-2 cells, we screened the CNE-2 cells by single clone selection. We stained the single clone cells respectively with Hoechst 33342, which is actively extruded by verapamil-sensitive ABC transpor- ters. The SP cells from representative single clone CNE- 2 ranged from 0.1% to 73.7% as estimated by FACS ana- lysis (Fig. 1A, B and 1C). In each case, by definition, the number of SP cells was decreased greatly by treatment with verapamil (Fig. 1 bottom panel). We could detect little SP cells in just a single clone cell (Fig. 1C, clone 2). Thus, most of the CNE-2 cancer cell clones con- tained a small or big component of SP cells. Our study is focus on whether microenvironment might have an effect on the percentage of SP cell in CNE-2, i.e, whether it play a role in the cancer stem cell pool, so the further experiments were mainly performed the human CNE-2 single clone (namely CNE-2-2) with little SP phenotype. We cultured the CNE-2-2 clone with conditioned medium and carried out FACS analysis for the presence of SP. As shown in Fig. 1D, the CNE-2-2 cell clone con- tained 9.8% SP cells after cultured with macrophage-like cells, whereas the control CNE-2-2 cells only contained 0.1% SP. The data therefore suggested that macrophage might be responsible for maintaining the CNE-2 cancer stem cell pool. Validation SP alteration in neuroblastoma SK-N-CH cells To further confirm the above phenomena, we selected another cell line, the human neuroblastoma cell line SK- N-CH in which we could barely detect the presence of