BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 rFDoiCgbulXarsaeton1mdavimceellnstuinbtayrpeeesxpressed by different SK-N-SH neu- DCX and vimentin are expressed by different SK-N-SH neurob- lastoma cell subtypes. The sets of figures A-E and F-J are representa- tive of a series of images taken from SK-N-SH cells cultured in control conditions. PC analysis (panels A and F) clearly shows the heterogeneity of the cell population. The triple IF for DCX (B), LIS1 (C) and TUB (D) shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co- expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel E). The triple IF for DCX (G), NF-68 (H) and VIM (I) shows that none of the DCX+ cells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCX- cells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel J). The NF-68 protein is expressed by all the cell subtypes. As expected, S-type cells are immunopositive for VIM (VIM+) that is totally absent (VIM-) in the N-type cells (Fig. 1). A double staining for DCX and VIM clearly shows an exclusive expression in the different cell types; in fact, these proteins never colocalize in any cell type observed. Among the cells with an intermediate morphology we identified at least three subpopulations: the first one shows immunoreactivity for DCX but not for VIM; the sec- ond one displays an opposite expression pattern of the two proteins; and the third one expresses neither DCX nor VIM (Fig. 1). NF-68 immunoreactivity is present in all the cells, even if the N-type shows a higher signal. Effects of exposure of SK-N-SH cells to retinoic acid As expected, 10 μM RA treatment induces a clear morpho- logical differentiation of SK-N-SH N-type cells shown by the extension of their neuritic processes, Some S-type cells appear more elongate, but do not acquire a completely different phenotype (Fig 2). In terms of DCX, after 2 (Fig. 2 panel M) or 4 days (data not shown) of RA exposure it is possible to observe a decreased stain in N-type cells while, in the same cell, the signal for VIM increases. After a longer (6 days) RA treat- ment, DCX immunoreactivity disappears from almost all of the N-type cells and it is replaced by VIM signal (Fig 2). The S-type cells remain DCX-, whereas VIM is always present even if the fluorescence signal is unchanged or slightly reduced (Fig. 2). A quantitative evaluation of the number DCX+ and VIM+ cells, regardless of their morphol- ogy, confirms that RA exposure leads to a decrease in the number of DCX+ cells and to an increase in that of VIM+ (Fig 2 panel K and L). Regarding NF-68, RA treated cells show a more intense signal, in particular the N-type cells that underwent differ- entiation (Fig. 2). After RA treatment, we also observe the same three sub- populations of I-type cells recognized in untreated cul- tures; moreover at this time interval, LIS1 immunostaining is apparently reduced, nevertheless it is still present in all cell types (data not shown). WB analysis clearly shows double immunoreactive bands for DCX in SK-N-SH (Fig. 3). This doublet resolves to a single band after calf intestine alkaline phosphatase (CIP) treatment, indicating that the slower-migrating species correspond to phosphorylated DCX (Fig. 3), as already described [14,25]. However, since we observed prelimi- narily that RA treatment reduced the amount of both DCX forms, and in order to better quantify the decrease of its expression, in the following experiments we run the sam- ples to get a single immunoreactive band for DCX, and Page 5 of 12 (page number not for citation purposes)