Sirt1 direct fluorescent screening assay kit

All the chemicals used in the experiments were of analytical grade. Acetonitrile (ACN) and methanol (MeOH) were HPLC gradient-grade (Merck KGaA (Darmstadt, Germany). Per-chloric (60%) and hydrochloric acid (37%) were from J.T. Baker (NJ, United States). Sigma-Aldrich Chemie (Sant Quentin Fallavier, France) provided Alcalase 2.4 L FG (CAS no. 9014-01-1), ortho-phthaldialdehyde reagent (OPA), l-glutathione, borate buffer, formic acid (for LC-MS LiChropur™), ammonium acetate, ammonium formate, phenyl isothiocyanate (PITC), triethylamine (TEA), pure standards for 19 amino acids, taurine, hypotaurine and homotaurine, trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), α-diphenyl-β- picrylhydrazyl (DPPH), potassium persulfate, Folin-Ciocalteu’s reagent, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO). The SIRT1 Direct Fluorescent Screening Assay Kit was from ABNOVA (Cat # KA1366, Taiwan).

SIRT1 activity modulation was assessed by using a Direct Fluorescent Screening Assay Kit (SIRT1 Direct Fluorescent Screening Assay Kit, Cat # KA1366, ABNOVA, Taiwan) following the instructions of the provider (Figures S1 and S2, Supplementary material). The formation of the final fluorescent product was detected using a Varioskan (Varioskan, Thermo Scientific, MA, USA) with an excitation wavelength of 360 nm and an emission wavelength of 465 nm.
For each EPH fraction, the % inhibition/activation was calculated following the Eq. (4), and the fold activation was calculated following the instructions of the provider by using the Eq. (5): $$\%Inhibition/Activation=\left[\frac{InitialActivityfluorescenc{e}_{\mathit{control}}-Samplefluorescence}{InitialActivityfluorescenc{e}_{\mathit{control}}}\right]\times 100$$ $$FoldActivation=\frac{Samplefluorescence}{InitialActivityfluorescenc{e}_{\mathit{control}}}$$ where “Initial Activity fluorescence _control” is the fluorescence obtained in wells with SIRT1 dissolved in assay buffer and solvent;
“Sample fluorescence” is the fluorescence from wells with SIRT1 dissolved in buffer assay and solvent plus samples.
To perform the Pearson´s correlation test and the principal component analysis, negative values of SIRT1 activity were scaled and normalized into a range of 0 and 1, according to Teknomo⁷⁵ .