Ab156065

SIRT1 enzymatic activity was measured using a commercial kit (ab156065; Abcam). According to the manufacturer's directions, fresh intestinal tissues were immunoprecipitated (Immunoprecipitation Kit; Proteintech, Chicago, IL, USA) with anti-SIRT1 antibody (Santa Cruz Biotechnology). Then, the reaction mixture containing fluoro-substrate peptide solution and protein A agarose beads was added, and NAD-dependent deacetylase activity was measured based on fluorescence intensity at 1-2 min intervals at Ex/Em = 350/460 nm in SpectraMax M5. The activity is presented as the relative value compared to the control group.

The mitochondrial global sirtuin activity was measured by SIRT-Glo™ Assay (#G6450; Promega, Madison, Wisconsin, USA) as recommended by the manufacturer. The activity of SIRT1 was measured in whole heart lysate using fluorometric assay (ab156065; Abcam, Cambridge, UK) according to the manufacturer´s recommendations.

CTSB activity was assayed fluorimetrically with Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride (60 μmol/L) at pH 7.4 and 37 °C as previously described.^{9 (link)} CTSS and SIRT1 activities were determined fluorimetrically using commercially available kits (ab65307 and ab156065 respectively, Abcam, Cambridge, UK) and following the manufacturer's instructions.

Hearts harvested from the sacrificed mice were arrested in 10% KCl, fixed in 4% paraformaldehyde overnight, and subsequently processed for paraffin embedding and sectioning into 5 μm slices. After hydration via a graded ethanol series, the sections were stained with hematoxylin and eosin (H&E) to outline the gross heart or picrosirius red (PSR) to determine the average collagen volume. Digital images of the slides were then captured by Photo Imaging System (H550L; Nikon, Tokyo, Japan). Each slide was blindly examined by two authors. The cross-sectional area and average collagen volume were determined by Image-Pro Plus 6.0 (Maryland, USA). In each group, 5 mice with 25 fields were used to count the cross-sectional area of cardiomyocytes. To evaluate the collagen volume, more than 30 fields in 5 mice per group were assessed.
Myocardial TNF-α levels were determined using an ELISA kit (#BMS607HS, Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. Sirt1 activity was determined using a commercial kit (ab156065) obtained from Abcam following the manufacturer's protocol.
We qualitatively analyzed myocardial apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining according to the manufacturer's instructions [9 (link), 17 (link)]. The images were quantified by Image-Pro Plus 6.0.

SIRT1 enzymatic activity was assessed by using commercial kits (ab156065) from Abcam plc. (Cambridge, UK) in accordance with the manufacturer’s instructions. First, assay buffer (50 mM TRIS-HCl, pH 8.0, 137 mM sodium chloride, 2.7 mM potassium chloride, 1 mM magnesium chloride, 1 mg/mL bovine serum albumin), SIRT1 enzyme, and either solvent (DMF) or different concentrations of elaiophylin were mixed with the co-substrate (NAD) for 45 min. Thereafter, the stop/developing solutions, containing a mixture of a developer, were added to the microplate and incubated for 30 min at 25 °C. The deacetylated peptide reacts with the developer and releases a fluorophore. The fluorophores in both assays were analyzed at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The inhibitory percentage of the samples on the SIRT1 enzyme activity was calculated as the ratio of fluorescent intensity between samples and vehicle control [21 (link)].

Oroxylin A (purity > 98% as determined by HPLC) was purchased from the National Institutes for Food and Drug Control (China). The specific Sirt1 inhibitors including EX527 (HY-15452) and nicotinamide (HY-B0150) were provided by MedChemExpress (Shanghai, China). DOX was provided by Sigma-Aldrich (St. Louis, MO, USA). The assay kits for Sirt1 activity (fluorometric, ab156065), NAD/NADH level (colorimetric, ab65348), protein kinase A (PKA, ab139435) activity, and cAMP level (competitive ELISA, ab138880) were provided by Abcam (Cambridge, UK).