*4.13. Mitochondrial Membrane Potential (MMP)*

Mitochondrial membrane potential was quantified using the fluorescent cationic dye tetramethylrhodaminemethylester (TMRM) following Correia et al.'s (2012) protocol with some modifications [52]. After cell treatments, the Krebs medium with calcium (1 mM CaCl2) and TMRM (250 nM) was added. A FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany) microplate reader was used to measure basal fluorescence activity at λ 549 nm excitation and λ 573 nm emission at 37 ◦C for 45 min. Then, the maximum fluorescence value was estimated after adding FCCP (6 mM) and oligomycin (0.25 mg/mL). Mitochondrial membrane potential (Δψm) is the result by subtracting fluorescence basal values from maximum fluorescence values. Results were expressed as % of the control.

#### *4.14. Secondary Metabolites Detection Using High Performance Liquid Chromatography*

HPLC analysis was used for secondary metabolites detection using the method described by de Paz et al. (2010) [53]. The HPLC instrumentation was an Agilent 1260 instrument (Agilent Technologies, CA, USA) equipped with a photodiode array detector (190–800 nm) and a reversed-phase Mediterranean Sea 18 column (150 mm × 4.6 mm, 3 μm particle size; Teknokroma, Barcelona, Spain). Running conditions included: a mobile gradient phase [1% orthophosphoric acid in milli-Q water (A)/methanol (B)]; a flow rate of 0.6 mL/min; a column temperature of 40 ◦C and a UV spectrum between 190 and 400 nm. Secondary metabolites of lichens were identified by comparing the retention time and UV absorption spectra with standard compounds (commercialized and isolated previously by our research team) and other lichen species [9,54].

### *4.15. Statistical Analysis*

All assays were measured in triplicate and data were expressed as mean ± SD. Statistical analysis was performed by SigmaPlot 11.0 using analysis of variance (ANOVA) and Tukey's post hoc test (5% significance level).

#### **5. Conclusions**

In conclusion, our findings provide evidence of the protective activity of methanol extracts obtained from cetrarioid clade against the neurotoxic effects of hydrogen peroxide in neuroblastoma cells. *D. arctica* and *V. pinastri* afford the highest protective effect. Future research should be aimed at studying the protective activity of isolated compounds from *D. arctica* and *V. pinastri*, delving into their mechanism of action.

**Author Contributions:** Conceptualization, I.U.-V., E.G.-B. and M.P.G.-S.; Methodology: I.U.-V.; Investigation, I.U.-V. and E.G.-B.; Formal analysis, I.U.-V. and E.G.-B.; Data Curation, I.U.-V. and E.G.-B.; Writing—Original Draft Preparation, I.U.-V. and E.G.-B.; Validation, E.G.-B.; Writing—Review & Editing, P.K.D. and M.P.G.-S., Supervision, E.G.-B. and M.P.G.-S., Project administration. P.K.D. and M.P.G.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Spanish Ministry of Science, Innovation and Universities (PID2019-105312GB-100) and the Santander-University Complutense of Madrid (PR87/19-22637). I. Ureña Vacas was supported by a grant CT42/18-CT43/18 from Complutense University of Madrid for predoctoral research.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

**Sample Availability:** Samples are not available from the authors.
