BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 FDiCguXrdeo5wn regulation is time dependent and irreversible DCX down regulation is time dependent and irreversible. WB representative image (A): lysates (50 μg/lane) of SK-N-SH control and RA treated cells at different time intervals (2, 4, 6, 6 plus 6 of withdrawal and 12 days) were analyzed by immunoblotting using an anti-DCX specific anti- body; membranes were reprobed with anti-tubulin as a loading control. Quantification of DCX protein amount by scanning densitometry (B): data are means ± standard error, N = 8, and are expressed as a percent- age of the control (100%). After 2 or 4 days of RA treatment DCX levels are significantly decreased, but to a lesser extent than after a 6 days expo- sure to RA. Lengthening SK-N-SH exposure to RA up to 12 days does not modify the magnitude of the DCX reduction observed after 6 days of treatment. Moreover, in cells treated with RA for 6 days and then grown for 6 additional days in medium without RA (6 days+6), DCX protein amount remains at low levels; single star = p < 0.05; double star = p < 0.01; C, control, RA, samples treated with RA 10 μM. SK cell line as DCX-expressing (DCX+) or DCX-not- expressing (DCX-) cells. In addition, we show that also in NB cells this protein colocalizes with tubulin, as already reported for mouse and human migrating neurons [13,28,29]. The data here reported indicate for the first time that LIS1, another MAP that may dimerize with DCX in migrating neurons [16], is expressed in all the cell types composing SK-N-SH cell line. It is well known that DCX-LIS1 interac- tion plays a role in nucleus-centrosome coupling during neuronal migration [17]. Therefore, it is possible to pos- tulate that LIS1-DCX interaction would play a role also in DCX+ NB cell nucleokinesis and, possibly, in NB cell motility. Moreover, here we report the presence of DCX in the two cell types (N- and I-type) that are indicated as the most malignant component of NB [2]. Actually, the results obtained by scratch-wound-healing assay and cell inva- siveness test, clearly indicate that in control conditions all the cells that move into the scratched area and invade the Matrigel-coated membrane are DCX+. It is noteworthy to RFAigutrreeat6ment reduces SK-N-SK neuroblastoma cell motility RA treatment reduces SK-N-SK neuroblastoma cell motility. (A) Representative PC images of untreated (Control) or 6 days RA treated cells which motility has been analyzed by scratch test. Images show the starting (0 hr after scratch) and the end (24 hr after scratch) point of the analysis. In control the distance between the borderlines becomes signifi- cantly shorter 24 hr after wounding, while it is still elevated in RA treated samples. (B) IF DCX analysis performed both in control and in RA treated cells 24 hr after wounding. In control samples the majority of invading cells are DCX+ (red). Even if the migration rate of the treated cells is reduced, few of them move in and invade the scratched area; all the cells still migrating after 6 days RA treatment are DCX+ (inset 1 and 2 of panel B are magnified in panel C). Nuclei are counterstained with DAPI (blue). say that all the DCX+ cells, the only that show motility and invasiveness, express LIS1; the solely expression of LIS1 in DCX- cells excludes a role of it in spontaneous motility. Of interest is the observation that VIM, an intermediate filament protein characteristic of non-neuronal and non- tumorigenic NB cell types [2,3], is present only in DCX- and not in DCX+ cells. This exclusive expression of DCX or VIM represents an additional parameter to identify differ- ent NB cell subtypes. Therefore, beside the well recogniz- able N-type and S-type cells, our results evidence at least three cell subtypes with a I-type morphology: DCX+, DCX- /VIM+ and, in a small amount, cells lacking both DCX and Page 9 of 12 (page number not for citation purposes)