*4.8. Quantitative Real-Time PCR*

Random primers and the SuperScript III kit (11752050, Invitrogen, Paisley, Scotland, UK) were used to reverse-transcribe 1 μg of total RNA into first-strand cDNA in a total volume of 20 μL according to the manufacturer's instructions. SYBR Green PCR Master Mix (a25778, Applied Biosystems, Carlsbad, CA, USA) was mixed with cDNA for quantitative real time polymerase chain reaction (qRT-PCR) using 0.3 μM forward and reverse oligonucleotide primers (Table 1). 7300 Real Time PCR System (Applied Biosystems, Carlsbad, CA, USA) was used for quantitative measure of gene expression. Cycling conditions were an initial denaturation at 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s 60 ◦C for 1 min. At the end, a dissociation curve was implemented from 60 to 95 ◦C to validate amplicon specificity. Relative quantification of gene expression was calculated by interpolation into a standard curve. All values were divided by the expression of the house keeping gene 18S rRNA.

**Table 1.** Primers used for qRT-PCR in this study.


The annealing temperature was 60 ◦C for all primers. 18S rRNA was used as a housekeeping gene.

#### *4.9. Data Analysis*

The data in the graphs are presented as the mean ± S.E.M, from at least five independent experiments. The normality of population and the homogeneity of variance were evaluated by the test of Kolmogorov–Smirnov with the correction of Lilliefors and the test of Levene, respectively. Then one- or two-way ANOVA tests followed by post hoc Tukey's test for multiple comparisons were used to compare the values. Statistical analysis was carried out with SPSS 18.0 software (IBM, Armonk, NY, USA). Significant differences were considered when *p* < 0.05.

**Author Contributions:** Conceptualization was agreed by J.T., I.M.L. and A.N., who also participated in the design of the project and analyzed the results. E.M.-P., N.R.-S. and E.G.-Á. performed the majority of the experiments; E.d.V. performed some of the immunocytochemistry assays and participated in data analysis; R.P. performed the qRT-PCR, participated in data analysis and in providing data for final figures; E.M-P. and J.T. did many of the imaging assays in the confocal microscope, took images and participated in data analysis; E.M.-P., A.N. and I.M.L. wrote the first draft of the manuscript that was further edited by all coauthors, who agreed with submission. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Instituto de Salud Carlos III through the project (PI15/00601) (co-funded by European Regional Development Fund/European Social Fund "Investing in your future"). This work was also funded in part by a grant (PI19/01805) from the Instituto de Salud Carlos III, co-funded by European Regional Development Fund (ERDF) "A way to build Europe" and by Fundación Rioja Salud. I.M.L. is supported by a Miguel Servet contract (CPII20/00029) from the Instituto de Salud Carlos III, co-funded by European Social fund (ESF) "Investing in your future".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


**Figure A1.** MTT assay in HOG cells treated with increasing concentrations of CLO (0.1–100 μM) during 24 h (**a**) and 48 h (**b**). Cell damage is represented as the percentage of viability versus control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared with control.

**Figure A2.** MTT assay in SH-SY5Y cells treated with increasing concentrations of CLO (0.1–100 μM) during 24 h (**a**) and 48 h (**b**). Cell damage is represented as the percentage of viability versus control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\*\* *p* < 0.001 compared with control.

**Figure A3.** MTT assay in HOG (**a**,**b**) and SH-SY5Y (**c**,**d**) cells treated with increasing concentrations of CPZ (50–1000 μM) and their respective vehicles for 24 h (**a**,**c**) or 48 h (**b**,**d**). Cell damage is represented as the percentage of viability versus control. Data are the mean ± S.E.M of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to control.
