BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 amino acids (NEAA), 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin (20 units/ml) and streptomycin (20 mg/ml), and were maintained at 37°C in a saturated humid atmosphere with 5% CO2. As they approached confluence, the cells were split following treatment with Trypsin-EDTA. The seeding density varied according to the type of experiment. DCX sequence analysis Genomic DNA from the SK-N-SH cell line was prepared using standard procedures [21]. Sequence analysis of the DCX gene coding regions was performed essentially as described previously [11]. Briefly, the 7 DCX coding exons (exons 1C-7; NCBI Ac. Nos. AJ005592-AJ005597) and their flanking intron sequences were amplified by PCR and directly sequenced using an automated sequencing apparatus (3100 Genetic Analyzer, Applied Biosystems, Foster City, CA). Treatment with Retinoic Acid All-trans RA (Sigma) was added to a final concentration of 10 μM, according to known pharmacological dosages used in phase I trials of RA administered orally to NB patients [22]. Moreover, it has been well established that addition of 10 μM RA to proliferating NB cells induces their differentiation [23]. To study RA effects on SK-N-SH cell line, cells were plated at a density of 5 × 105cells/10 cm diameter Petri dish for WB at a density of 5 × 103cells/ 13 mm diameter well for IF or alternatively at a density of 2 × 104cells/13 mm diameter well for the scratch wound assay, and grown in MEM medium supplemented as described above. RA was first dissolved in absolute etha- nol and then supplemented with DMSO to achieve a con- centration of 10-3 M in ethanol-DMSO (87.5%–12.5%). This stock solution was kept at -20°C. For each experi- ment RA was diluted at the desired concentration directly into the growth medium. Cells cultured in medium con- taining 0.1% of ethanol-DMSO (87.5%–12.5%) were used as control. The 10 μM RA treatment started on the second day after plating, concomitantly with the medium replacement. Cells were fed every 48 hr with control, or RA- containing fresh medium and the treatment was stopped 2, 4, 6 or 12 days after the first addition of RA. In order to investigate whether the effect of RA on DCX pro- tein expression was reversible, we treated SK-N-SH cells with RA for 6 days; afterwards the cells were grown in con- trol medium for 6 additional days. Antibodies For both IF and WB the primary antibodies used were: anti-DCX (C-18 sc-8066; 1:500) and anti-LIS1 (H-300 sc- 15319; 1:200) from Santa Cruz Biotechnologies, anti- vimentin (1:10,000) and anti-neurofilaments-68 (1:1,000) from Chemicon International and anti-α-tubu- lin (1:500) from Sigma. Immunofluorescence analysis Cells were grown on 13 mm diameter coverslips, fixed in 4% paraformaldehyde (PFA), permeabilized in 0.2% Tri- ton X-100 and then, the unspecific sites were blocked in 10% normal donkey serum (Sigma) in PBS. Subsequently, cells were incubated with the primary antibodies for 1 hr at room temperature and then rinsed with PBS. For triple- label IF, Cy3-conjugated anti-goat (for anti-DCX anti- body), Cy2-conjugated anti-rabbit (for anti-LIS1, or alter- natively for anti-neurofilaments-68 antibody) or AMCA- conjugated anti-mouse (for anti-α-tubulin or alternatively anti-vimentin antibody) secondary antibodies (Jackson ImmunoResearch) were used (1 hr at room temperature). The manufacturer guarantees the use of these secondary antibodies for multiple labelling procedures, since they are purified against cross-reactivity to other species. Nuclei were stained with DAPI (Sigma-Aldrich). Finally, the cells were washed in PBS before mounting using Mowiol 40–88 (Sigma-Aldrich). Western blot analysis Cells were lysed with modified RIPA buffer (TRIS-HCl 50 mM pH7.4, NaCl 150 mM, Tryton X-100 0.1%, Na-deox- ycholate, EDTA 0.1 M, SDS 1%) containing protease (ABESF, leupeptin and pepstatin) and phosphatase inhib- itors (NaF 1 mM, Na3VO4 1 mM). For alkaline phos- phatase treatment, cells were lysed with the aforementioned RIPA cell lysis buffer without phos- phatase inhibitors (1 mM NaF and 1 mM Na3VO4) and then the cell lysates were incubated at 37°C for 30 min- utes with calf intestinal alkaline phosphatase (CIP, 1 unit/ μg of protein; Amersham Biosciences). The total protein content for each sample was measured using the Bradford's method. 50–70 μg of proteins were separated on SDS-12% PAGE under reducing conditions and transferred onto nitrocellulose membranes. After blocking for 45 minutes at room temperature in 5% non- fat-milk in PBS, membranes were probed overnight at 4°C, with the primary antibodies diluted in 5% milk-PBS; they were subsequently washed with PBS and incubated with secondary antibodies coupled to horseradish peroxi- dase. The secondary antibodies used were HRP-conju- gated- anti-goat (for anti-DCX antibody), anti-rabbit (for anti-LIS1 or for anti-neurofilamente-68 antibody), and anti-mouse (for anti-α-tubulin or anti-vimentin anti- body) from Santa Cruz Biotechnologies. Detection and visualization of protein-bound antibodies was achieved using an enhanced chemiluminescence WB detection kit (ECL Plus, Amersham Biosciences). To quantify the inten- sity of the bands, membranes were scanned and analysed with the NIH Image software. All data are normalized against tubulin and expressed as percentage of the control. Page 3 of 12 (page number not for citation purposes)