**4. Materials and Methods**

#### *4.1. Patients*

The Local Ethics Committee approved the present study. All experiments were performed on twelve patients (six males and six females) affected by moderate psoriasis (PASI = 12.4 ± 0.5) with a mean age of 41.4 ± 7.6 years and with a mean disease duration of 14.1 years (from 8 to 25 years). The demographic and clinical data for each patient are summarized in Table 1. Seven healthy controls (four males and three females), matched for age and body mass index, were also enrolled in the study. No subject was subjected to any systemic therapy before or during the study, or had a history of any disease, e.g., diabetes mellitus and atherosclerosis, which might affect blood redox status. All subjects provided signed informed consent. The study was carried out according to the Helsinki Declaration.


**Table 1.** Demographic and clinical data of psoriatic patients involved in the study.

### *4.2. Fibroblast Isolation and Setting Up of Cell Cultures*

Lesional skin punch biopsies from twelve patients affected by plaque psoriasis and from seven healthy controls were used to obtain primary fibroblasts cell cultures. Briefly, skin biopsies were incubated with dispase II (2 U/mL, Sigma-Aldrich, Milan, Italy) and the derma was digested with collagenase (3 mg/mL, Sigma-Aldrich) for 45 min at 37 ◦C. Then, cells were filtered through a 70 μm filter to remove debris. Cells were cultured in DMEM medium (Sigma-Aldrich) with 10% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). Fibroblasts were characterized by a high Vimentin (Sigma-Aldrich) expression by FACS and confocal microscopy analyses. Cells up to the fourth passage were used for experiments.

### *4.3. Preparation of Cell Homogenates*

After trypsinization, fibroblasts (1 × 106) were resuspended in 100 <sup>μ</sup>L of lysis buffer (20 mM Tris-HCl pH8, 1% Triton X-100, 10% (*v*/*v*) glycerol, 137 mM NaCl, 2 mM EDTA and 6 M urea supplemented with 0.2 mM PMSF, 10 mg/mL leupeptin + aprotinin). Samples were then twice sonicated in ice for 5 s, centrifuged at 14,000× *g* for 10 min at 4 ◦C, and the supernatant collected. Protein concentration was determined according to the Bradford method [65].

#### *4.4. Western Blot Analysis of SIRT1*

SIRT1 expression levels were assessed by Western blot analysis. Briefly, equal amounts of homogenates (50 μg) were separated on 4–12% SDS-PAGE gels (Criterion XT, Bio-Rad Laboratories, Milan, Italy). After blotting into PVDF Hybond membranes and incubation overnight at 4 ◦C with (rabbit) anti-SIRT1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), membranes were washed and incubated for 1 h with peroxidase-conjugated secondary antibody. Then, bands detected with a SuperSignal West Dura (Pierce, Rockford, IL, USA) were quantified using Quantity-One software (Bio-Rad, Milan, Italy) [66]. SIRT1 expression levels were calculated as ratios between the densitometry of the corresponding band and the loading control (GAPDH).
