**2. Results**

## *2.1. Apo D Expression in HOG and SH-SY5Y Cells in Response to Cuprizone Treatment*

Taking advantage of our experience in the CPZ-induced cell model, some previous results showing that CPZ was able to induce cytotoxic damage mediated by a mitochondrial dysfunction in the HOG and SH-SY5Y cell lines (data not shown), and the findings reported in this work (see next figures), we aimed to analyze the potential effect of CPZ on Apo D expression in these oligodendroglioma and neuroblastoma cell lines by qRT-PCR and immunocytochemistry. As shown in Figure 1, the analysis of Apo D gene expression (Figure 1a) and the immunosignal quantification (Figure 1b–d) revealed that CPZ induced changes in Apo D expression and, interestingly, only at the highest concentration (1000 μM) and at 48 h of treatment. In fact, a constant and almost invariable fluorescence signal was observed in control and treated cells (Figure 1b).

**Figure 1.** Relative Apo D gene expression in HOG cells treated with 0–1000 μM of CPZ following 24 h. Data represent the quotient between the gene and the expression of the housekeeping gene 18S rRNA. Bars represent the mean ± SEM of all measurements (*n* = 6–8) (**a**). Representative fluorescence microscopy images of Apo D levels in HOG cells treated or not with 1000 μM of CPZ during 24 and 48 h. 40× magnification (**b**). Densitometric quantification of Apo D immunocytochemical signal after 24 (**c**) and 48 h (**d**) of treatment with increasing concentrations of CPZ (50–1000 μM) in HOG cells (*n* = 6). Bars represent mean density per cell in a 40× field ± SEM (over control). Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared with control.

As expected in the case of SH-SY5Y neuroblastoma cells, which according to previous studies show a negligible expression of Apo D [52], we found that these cells exhibited a very scarce endogenous expression of Apo D only detected by immunocytochemistry, and that CPZ did not influence the apolipoprotein synthesis as observed in the images (Figure 2a) and the immunocytochemical quantification (Figure 2b,c).

**Figure 2.** Representative fluorescence microscopy images of Apo D levels in SH-SY5Y cells treated or not with 1000 μM of CPZ during 24 and 48 h. 40× magnification (**a**). Densitometric quantification of Apo D immunocytochemical signal after 24 (**b**) and 48 h (**c**) of treatment with increasing concentrations of CPZ (50–1000 μM) in SH-SY5Y cells (*n* = 6). Bars represent mean density per cell in a 40× field ± SEM (% versus control).
