*4.10. Mitochondrial Number*

MitoTracker Deep Red 633 (Life Technologies, Carlsbad, CA, USA) was used to stain mitochondria by confocal microscopy as previously described [21]. Mitochondrial number was also monitored by flow cytometry. Cells were incubated with MitoTracker Deep Red 633 (200 nM) for 20 min at 37 ◦C and immediately analysed by a FACSCanto flow cytometer (Becton-Dickinson).

#### *4.11. Mitochondrial Permeability Transition Pore Opening*

The fluorescent calcein-AM probe was used to assess mitochondrial permeability, an indicator of mitochondrial dysfunction and early apoptosis, as described by Petronilli et al. [24], albeit with minor modifications [71]. Calcein-AM, after entering the cell and following deesterification, emits fluorescence. Cell cobalt chloride co-loading quenches cell fluorescence except for mitochondria where cobalt cannot enter (living cells). In contrast, in the case of mitochondrial permeability transition pore opening (mPTP), cobalt enters mitochondria and quenches calcein fluorescence (apoptotic cells). Thus, decreased mitochondrial calcein fluorescence represents a measure of the extent of mPTP induction. Mitochondrial permeability transition pore opening was monitored by confocal microscopy and FACS analysis [71].

#### *4.12. Mitochondrial Membrane Potential*

Tetramethylrhodamine methyl ester perchlorate (TMRM) was used to assess the mitochondrial membrane potential. For confocal microscope analysis, cells were cultured on glass cover slips and loaded for 20 min at 37 ◦C with 100 nM TMRM (Life Technologies, Carlsbad, CA, USA). After fixing, cells were analyzed using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany). Mitochondrial membrane potential was also quantified by flow cytometry. Cells were incubated for 20 min at 37 ◦C with TMRM (100 nM) in DMEM and analyzed using a FACSCanto flow cytometer (Becton-Dickinson).

#### *4.13. Determination of Caspase Activity*

Caspase-3 and caspase-9 activity were analysed by confocal microscopy and FACS analysis. Fibroblasts were loaded with FAM-FLICA™ Caspases solution (Caspase FLICA kit FAM-DEVD-FMK) for 1 h at 37 ◦C, washed twice with PBS and analysed by a Leica TCS SP5 confocal laser scanning microscope and FACSCanto flow cytometer (Becton-Dickinson) [72]. In another set of experiments, aimed to assess the role of ERK, p38 and JNK signalling pathways, cells were treated with 10 μM SP600125 (specific JNK inhibitor), 10 μM PD98059 (MEK inhibitor) or 1 μM SIRT1 inhibitor for 3 h prior (or not) to SRT1720 treatment.

#### *4.14. SIRT1 RNA Interference (RNAi) Experiments*

Fibroblasts from healthy subjects were cultured in complete medium without antibiotics for 2 days. Cells were seeded into a six-well plate. Then, 8 μl of LipofectamineTM LTX and 3 μL PLUSTM Reagent (Life Technologies, Carlsbad, CA, USA) were diluted in 90 μL of culture medium. Subsequently, 12 μL SIRT1 siRNA (siRNA for SIRT1-sc-40986- from Santa Cruz Biotechnology) was mixed with the medium containing Lipofectamine together with PLUS reagent and incubated for 30 min at RT for complex formation. Finally, cells were incubated with a final SIRT1 siRNA concentration of 100 nM. After 48 h, SIRT1 protein expression was determined by Western blot. To study the possible role of SIRT1 in the oxidative-mediated cell injury, untreated and SIRT1 RNAi-treated fibroblasts obtained from healthy subjects were challenged for 3 h with 500 μM H2O2. ROS production, lipid peroxidation, caspase-3 activity and mitochondrial membrane polarization were evaluated by confocal microscope analysis.

#### *4.15. Assessment of MAPK Activity by FACS Analysis*

Fibroblasts were fixed and permeabilized using the BD Cytofix/Cytoperm buffer (Becton-Dickinson) following the manufacturer's instructions. Anti-Phospho-p38 MAPK (Thr180/Tyr182)

(28B10) Mouse mAb (Alexa Fluor® 488 Conjugate), anti-Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb (PE Conjugate), and anti-Phospho-p44/42 MAPK (Erk1/2) (T hr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) were used at 1:50 dilution for 1 h at RT according to manufacturer's instructions.
