Microson

Cells were lysed in IP Lysis/Wash buffer (Thermo Fisher Scientific) supplemented with 5% (vol/vol) Protease Mixture Inhibitor (MilliporeSigma) and 1 mM phenylmethanesulfonyl (MilliporeSigma). After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged at 18,000g for 10 minutes. The supernatant containing proteins was collected and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Immunoblotting analysis was carried out as previously described (8 (link)). Antibodies against Actin (MilliporeSigma, A2066; dilution 1:4000), DOT1L (Cell Signaling, 77087; dilution 1:1000), HIF1A (Abcam, ab82832; dilution 1:1000), total H3 (Abcam, ab1791; dilution 1:10,000), and H3K79me2 (Abcam, ab3594; dilution 1:1000) were used following the manufacturer’s instructions. The blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Fisher Scientific).

NIH3T3 and MEF cells were stimulated with 100 nM dexamethasone containing medium at each time point. Cells were fixed in 1× PBS containing 0.5% formaldehyde. Glycine was added to a final concentration of 0.125 M, and the incubation was continued for an additional 15 min. After washing the samples with ice-cold phosphate-buffered saline, the samples were homogenized in 1 mL of ice-cold homogenize buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and protease inhibitors cocktail) and centrifuged (15,000× g, 4°C, 5 min). The pellets were suspended in nucleus lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, protease inhibitors) and sonicated 20 times for 30 s each time at intervals of 60 s with a Bioruptor (Diagenode, Inc.) or sonicated 10 times for 10 s each time at intervals of 50 s with a MICROSON (Misonix, Inc.) for brain samples. The samples were centrifuged at 15,000 rpm at 4°C for 5 min. Supernatants were diluted 10-fold in ChIP dilution buffer (50 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, protease inhibitor).

The deacetylase activity of SIRT1 was determined by using a SIRT1 Activity Assay Kit (Abcam, ab156065) as recommended by the manufacturer. Briefly, cells were washed with cold PBS, lysed in IP Lysis/Wash buffer (Thermo Fisher) and incubated on ice for 5 min. After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged 10 min at 13,000g, and supernatant containing proteins was collected. The protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Cell lysates (200 μg) were incubated with anti-SIRT1 antibody (10 μg) (Abcam, ab7343) for 3 h at 4 °C and then with protein A Agarose beads for the next 1.5 h. Precipitates were incubated with Fluoro-Substrate Peptide Solution, NAD⁺ and SIRT1 Assay Buffer. Fluorescence intensity was then measured using a microtitre plate fluorometer with excitation at 360 nm and emission at 460 nm.