*2.1. Reagents*

All common reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), Extrasynthèse (Genay, France), GE Healthcare (Little Chalfont, Great Britain), Santa Cruz Biotechnologies (Santa Cruz, CA, USA), Millipore (Bedford, MA, USA), Abcam (Cambridge, UK), Euroclone (Milan, Italy), Thermo Scientific (Waltham, MA, USA), Bioassay Systems (Hayward, CA, USA), Promega (Madison, WI, USA), and Invitrogen (Carlsbad, CA, USA), unless di fferently specified in the text.

The following reagents were purchased from Sigma-Aldrich: Ham's F12 Coon's modification medium, l-glutamine, dimethyl sulfoxide (DMSO), BSO, trypsin, bovine serum albumin, Tris/HCl, Triton X100, NaCl, NaF, ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA), β-glycerophosphate, human sirtuin type 1 (SIRT1) small interfering RNA (siRNA), Universal Negative Control #1, Alizarin Red S, phenolic reference standards ellagic acid (assay HPLC ≥ 95%) for hydroxybenzoic acids, (+)-catechin hydrate (assay HPLC ≥ 96.0%) for flavan-3-ols), acetonitrile HPLC grade (assay 99.9%), formic acid for (HPLC assay 98–100%), ascorbic acid, dexamethasone, paraformaldehyde, cetylpyridinium chloride, Folin–Ciocalteu reagent, NaCl/Pi.

The following reagents were purchased from Extrasynthèse: anthocyanin reference standard cyanidin 3-glucoside chloride (assay HPLC ≥ 96%), flavonol reference standard quercetin 3- *O*-glucoside (assay HPLC ≥ 99%), hydroxycinnamic acid reference standard 3- *O*-ca ffeoyl quinic acid (chlorogenic acid, assay HPLC ≥ 99%).

The following reagents were purchased from GE Healthcare: penicillin/streptomycin 100× solution, phosphate-bu ffered saline (PBS), polyvinylidene fluoride (PVDF) membrane, enhanced chemiluminescence (ECL) Western Blotting Detection Reagent kit.

The following reagents were purchased from Santa Cruz Biotechnologies: EX527, Protein A/G PLUS-Agarose, anti-Runt-related transcription factor 2 (RUNX-2), anti-phospho-tyrosine, anti-histone H3.

The following reagents were purchased from Milipore: Milli-Q water, Cytobuster Protein Extraction Reagent.

The following reagents were purchased from Abcam: SIRT1 ELISA kit, anti-histone H3.

The following reagent was purchased from Euroclone: fetal bovine serum South American origin.

The following reagent was purchased from Thermo Scientific: Pierce bicinchoninic acid (BCA) protein assay kit.

The following reagent was purchased from Bioassay Systems: QuantiFluo Alkaline Phosphatase Assay Kit.

The following reagent was purchased from Promega: CellTiter-Glo Luminescent Cell Viability Assay.

The following reagents were purchased from Invitrogen: lipofectamine RNAiMAXTM, 2,7-dichlorodihydrofluorescein diacetate.

#### *2.2. Preparation of Blueberry Juice and Determination of Total Soluble Polyphenols*

BBs, harvested in August 2018/2019 in Tuscany Apennines and supplied by IL BAGGIOLO S.R.L. (Abetone, Pt, Italy) and DANTI GIAMPIERO S.R.L. (Cutigliano, Pt, Italy), were frozen freshly picked in aliquots of 100 g each and homogenized in a refrigerated Waring Blender to prepare BJ. Insoluble

particles were removed by filtration under vacuum and centrifuged at 20,000× *g* for 10 min. Aliquots of BJ were stored at −20 ◦C until use. The total soluble polyphenol (TSP) fraction of BJ was quantified with Folin–Ciocalteu reagent using gallic acid as the standard as described in our previous work [33] and via the HPLC method reported below. TSP concentration in BJ obtained from 100 g of BB fresh weight was expressed as mg/100 mL ± SD and the values measured by Folin–Ciocalteu assay or HPLC method were 169.5 ± 19.4 and 158.8 ± 12.3, respectively.

#### *2.3. HPLC-PDA-MS Analysis of Phenolic Compounds*

The identification of phenolic compounds was performed using a Waters Alliance 2695 coupled online with a Waters 2996 photodiode array detector, and with a Quattro micro mass spectrometry detector with an electrospray interface. Separations were performed on a C18 reversed-phase Gemini Phenomenex (150 × 3 mm, 5 μm particle size) with a mobile phase flow rate of 0.4 mL·min−1. The mobile phase consisted of (A) H2O containing 5% formic acid and (B) MeCN. A gradient elution program was applied as follows: 0–1.0 min held on 8% B, 1.0–16.0 min linear gradient to 15% B, 16.0–28.0 min linear gradient 50% B, 28.0–36.0 min linear gradient to 95% B, then in 1 min to the initial (starting) condition, and held 8 min for re-equilibration. The total run time was 45 min. The sample was diluted 1:10 (*v*/*v*) with 8% B and 92% A, with an injection volume of 10 μL. Determination of phenolic compounds was performed using two detectors online: a photodiode array UV detector, followed by a single quadrupole mass spectrometry detector. The photodiode array scanned the samples at λmax 270, 320, 360, and 520 nm. The mass spectrometer detector was optimized to the following conditions: capillary voltage 3.20 kV, source block temperature 125 ◦C, and desolvation temperature 350 ◦C, operating in electrospray positive mode, detection range 100–1000 Da with total ion count extracting acquisition. The cone voltage was 32 V, the extractor lens was 3 V, and the cone and desolvation gas flows were 20 and 320 L·h−1, respectively. Phenolic compound identification in the sample was carried out by comparing UV absorption spectra and mass spectra of each compound with those reported in the literature [42]. The quantification of polyphenols was calculated using the method of an external standard. Each standard was freshly prepared up to 300 μg/mL concentration and injected three times to obtain its calibration curve. Quantification was obtained as total content of each polyphenol group. Quantification of total constituents of each class of flavonoids was carried out using single anthocyanin, flavonol, and flavan-3-ol standards, namely, cyanidin-3-glucoside, quercetin-3-glucoside, and (+)-catechin equivalents, respectively. The values were expressed as gallic acid equivalent.

#### *2.4. Cell Cultures, Treatments, Osteogenic Di*ff*erentiation, and Cellular Viability*

Osteoblast-like SaOS-2 cells were cultured in Ham's F12 Coon's modification medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 72 mg/L penicillin, and 100 mg/mL streptomycin (growth medium, GM), and incubated at 37 ◦C in a 5% CO2 humidified atmosphere with 20% oxygen. For the experiments of osteogenic differentiation, when SaOS-2 cells reached 70–80% confluence, GM was changed with osteogenic medium (OM) to induce osteogenic differentiation. OM was the growth medium supplemented with 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate. Then, 40 μM BSO, or BJ containing 7.5 or 15 μg·mL−1 TSP, or BJ + BSO, or BJ + 10 μM EX527, or BJ + BSO + 10 μM EX527 were added or not to GM for 24 h (day 1) before exchanging it for OM, containing or not the before mentioned compounds, to stimulate the differentiation process. The OM was refreshed twice a week for the whole study period, BSO was added for only two days after the beginning of differentiation and BJ or EX527 were added to the OM at each change from the beginning of the differentiation process for the whole study period.

Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay, according to the manufacturer's instructions.

In some experiments, the cells were transiently transfected with 75 nM human SIRT1 siRNA corresponding to two DNA target sequences of human SIRT1 (5-GUGUCAUGGUUCCUUUGCA[dT][dT]-3 accession number SASI\_Hs01\_00153666; 5UGCAAAGGAACCAUGACAC[dT][dT]-3, accession number SASI\_Hs01\_00153666 6\_AS) or scrambled siRNA (Scr siRNA) (Universal Negative Control #1), using lipofectamine RNAiMAXTM, according to the manufacturer's instructions. The ability of SIRT1 siRNA to silence SIRT1 expression levels of about 50% was checked in control cells transfected for 24 h in GM and for other 48 h in OM. Additionally, 0.008% DMSO was present in experiments with EX527 in all conditions.

## *2.5. Determination of Intracellular ROS*

The cell-permeant probe, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA), was added in the culture medium of SaOS-2 seeded in 12-well plates one hour before the end of the various treatments performed as written above. The probe after deacetylation by esterases is rapidly oxidized to a highly fluorescent compound in the presence of ROS. After PBS washing, adherent cells were lysed in radioimmunoprecipitation assay RIPA bu ffer (50 mM Tris/HCL pH 7.5, 1% Triton X-100, 150 mM NaCl, 100 mM NaF, 2 mM EGTA, phosphatase, and protease inhibitor cocktail), centrifuged at 20,000× *g* (ALC PK121R, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min, and the intracellular levels of ROS were measured by florescence analysis at 510 nm. The normalization of the data was obtained by using total proteins, and the values were expressed as percentages with respect to the controls.