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Activity was monitored by the formation of a fluorescent product (coumarin) generated by a two-step coupled reaction that involved deacetylation of substrate by SIRT-1 followed by secondary release of the fluorophore

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Afterwards, cells were lysed by three freeze and thaw cycles in liquid nitrogen and a 37 C waterbath, followed by addition of 900 L resuspension buffer (10 mM MgCl 2 , 10 mM ZnCl 2 , 1 M Tris-HCl, pH 8.0) and warming the samples to 28 C

( B ) Flow cytometry of SSP4 stained HeLa cells treated with RSL3 (5 M) for 4 hours