*4.10. TBARS Assay*

Lipid peroxidation was determined by performing a TBARS assay [49]. After treatments, cell pellets were stored at 80 ◦C. On the day of the experiment, pellets were defrosted at room temperature and were mixed with TBA-TCA-HCl. This mixture was boiled at 100 ◦C for 10 min. Samples were placed on ice to stop the reaction. Then, samples were centrifuged at 4 ◦C (3000 rpm, 10 min) and supernatants were added into 96-well plates to measure absorbance at 530 nm using a SPECTROstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Results were expressed as a percentage of TBARS (100% of control).

#### *4.11. Calcium Cytosolic Quantification*

Calcium cytosolic was quantified using Indo-1/AM as a cell-permeant dye [50]. After treatments, a Krebs medium containing Indo-1/AM dye (3 mM) and calcium (1mM CaCl2) was added to cells for 45 min at 37 ◦C. Then, the medium was removed, and cells were incubated with a dye-free Krebs medium for 15 min at 37 ◦C in the dark. Fluorescence was recorded in a microplate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany) at 350 nm excitation wavelength and at 410 nm emission wavelength. The formula for cytosolic calcium concentration was [Ca2+]i = *Kd* × [*<sup>F</sup>* − *Fmin*]/[*Fmax* − *<sup>F</sup>*], where Kd is the dissociation constant for Indo-1; F is the fluorescence signal for samples; Fmax is the maximum fluorescence signal after ionomycin addition and *Fmin* is calculated using this formula: *Fmin* = AF + 1/12 × (*Fmax*-AF), AF being the minimum fluorescence after adding MnCl2.

### *4.12. Mitochondrial Calcium Quantification*

Mitochondrial calcium was quantified using Rhod-2/AM as a fluorescent cationic probe [51]. After treatments, cells were incubated in a Krebs medium containing 0.1% BSA, 1 mM calcium and 10 mM Rhod-2/AM during 40 min at 37 ◦C. Then, cells were maintained in dye-free Krebs medium cells in this medium for 30 min at 37 ◦C in the dark. Basal fluorescence intensity was recorded using a microplate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany) at λ 552 nm excitation wavelength and λ 581 nm emission wavelength for 5 min at 37 ◦C. Then, maximum fluorescence was measured for 15 min after adding calcium ionophore A23187 (5 μM) in the same conditions as described above. Mitochondrial calcium levels were the ratio between fluorescence measures before and after ionophore addition. Results were expressed relative to control.
