Sirt1 activity was measured using the Sirt1 fluorimetric drug discovery kit (BML-AK555, ENZO Life Sciences International Inc, Plymouth Meeting, PA, USA).
For inhibition of human recombinant HDAC8, the HDAC8 Colorimetric/Fluorimetric Activity Assay/Drug Discovery Kit (cat no. AK-508; Biomol) was used.
The HDAC-1 and 2 calorimetric assay drug discovery kit is based on the unique Color de lys substrate and developer combination.
Lysine deacetylase activity of Sir2 and other enzymes was evaluated using a fluorogenic peptide substrate (Fluor-de-Lys green HDAC drug discovery kit: BML-AK530-0001, Enzo Life Sciences).
The HDAC-8 fluorimetric drug discovery kit is based on the unique fluoro de lys HDAC-8 substrate and developer combination.
Cytokine release of fully differentiated cells was measured using a 4-plex Meso Scale Discovery kit (IL-6, IL-8, TNFα and IL-1β) and singleplex kits (MCP-1 and IP-10; Meso Scale Discovery®).
DPP-4 activity was determined in conditioned medium or cell extracts from cell cultures using the DPP-4 drug discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA) with the Gly-Pro-para-nitroaniline (pNA) chromogenic substrate, according to the manufacturer's instructions.
Plasma was separated by centrifugation of blood at 2000 g for 5 minutes and total GLP-1 levels assayed using a two-site microtitre plate-based immunoassay with electrochemical luminescence detection (Meso Scale Discovery kit, Gaithersburg, MD).
SIRT1 activity levels were determined using recombinant SIRT1 (b; SIRT1 was incubated with AS101 or PBS at different concentrations for 1 hour. Activity was measured using the SIRT1 Fluorometric Activity Assay/Drug Discovery Kit Biomol (Exter, United Kingdom).
To investigate this further, we measured the levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) (two key cytokines associated with SASP) in the A2780, PEO1, and PEO4 cells using V-PLEX Human Proinflammatory (MeScaleale Discovery) kit (Fig. 3F and G).
