*4.5. Determination of Cellular SIRT1 Activity*

SIRT1 activity was determined according to the method described by Fulco et al. [67] with some modifications. SIRT1 activity was determined by the SIRT1 Direct Fluorescent Screening Assay Kit (Cayman, Ann Arbor, MI, USA) as previously reported [22]. The fluorescence was detected using a Perkin-Elmer (Milan, Italy), LS 55 luminescence spectrometer (ex: 360 nm; em: 460 nm).

#### *4.6. Cell Treatments*

Fibroblasts from psoriatic patients or controls were grown for 24 h in the presence of 10 μM SRT1720 (a selective small molecule activator of SIRT1).

In another set of experiments, cells were treated with 10 μM p38 kinase inhibitor (SB203580), 10 μM JNK inhibitor (SP600125), 10 μM MEK inhibitor (PD98059), and 1 μM specific SIRT1 inhibitor (6-Chloro-2,3,4,9-tetrahydro-1*H*-Carbazole-1-carboxamide) for 3 h. All reagents were purchased from Sigma at the highest purity available.

#### *4.7. Determination of Total Antioxidant Capacity (TAC)*

As previously reported, intracellular TAC was measured in cell lysates by a chemiluminescent assay using an Abel Antioxidant Test Kit (Knight Scientific Limited, Plymouth, UK) [68]. The protein content was measured by the Bradford method [65].

#### *4.8. Determination of Lipid Peroxidation*

8-Isoprostane levels (lipid peroxidation index) were measured in cell lysates using the 8-isoprostane EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA), following the manufacturer's instructions. Moreover, lipid peroxidation was investigated by confocal microscopy using BODIPY 581/591 C11 (Life Technologies, Carlsbad, CA, USA) [69]. The fluorescent probe BODIPY 581/591 C11 shifts its fluorescence from red to green in the presence of oxidizing agents. Fluorescence was detected using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) using a Leica Plan Apo 63X oil immersion objective and then projected as a single composite image by superimposition. Lipid peroxidation was also quantified by FACS analysis. Cells were incubated in DMEM with BODIPY 581/591 C11 (2 μM) for 30 min at 37 ◦C [70] and analyzed using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA).

#### *4.9. Assessment of Intracellular ROS, NO Production and Mitochondrial Superoxide*

After seeding on glass cover slips, fibroblasts were loaded for 15 min at 37 ◦C with the following fluorescent probes: MitoSOX (mitochondrial superoxide-specific probe, 3 μM), DAR-1 (NO probe, 1 μM) and H2DCF-DA (ROS production probe, 1 μM) all purchased from Life Technologies, Carlsbad, CA, USA. Cells were fixed in 2.0% buffered paraformaldehyde for 10 min at RT, and the fluorescence was detected by a Leica TCS SP5 confocal scanning microscope (Mannheim, Germany). Mitochondrial superoxide, NO and ROS generation were also monitored using the same fluorescent probes (MitoSOX: 0.5 μM; H2DCF-DA: 1 μM; DAR-1: 1 μM) by FACSCanto flow cytometer (Becton-Dickinson) [70].
