Sirt6

Cells were harvested at 80% confluency after 48 h with or without cumate. Trypsin was used to remove cells from the plate and then neutralized with PBS containing FBS and spun down to collect the cell pellet. The cell pellet was washed with cold PBS 1x prior to using Abcam Ab500 ChIP Kit. H3K9ac (Abcam ab4441), H3K18ac (Abcam ab1191), and SIRT6 (Cell Signaling #12486) antibodies were used. The manufacturer's protocol was used with the following considerations: Fixation time = 10 min at 37°C, Sonication settings (Qsonica Instrument) = 12.5 min, 15 s on/off, 30% output, 4°. qPCR primers from (Kawahara et al, 2009 (link); Tasselli et al, 2016 (link)) were used.

Cells and reactions were collected using a 2x Laemmli solution and incubated on ice for 15 min, during which time the samples were passed through a large gauge needle several times and vortexed every 5 min. Samples were then spun at 14,000 RPM to remove debris, and the supernatant was transferred to a new tube. Samples were heated in boiling water for 20 min before being centrifuged at 14,000 RPM for 1 min and loaded into a BioRad Criterion 4–20% gel. After transfer to PDVF membrane and blocked (5% dehydrated milk) for 2 h at RT, membranes were incubated overnight antibodies in 2.5% blocking buffer at 4°C. Membranes were washed 3x with TBST for 10 min each before secondary antibody in 1x TBST was added for 2 h incubation at RT. Membranes were washed 3x with TBST for 10 min and then imaged.
The following antibodies were used: H3 (Abcam ab500)‐1:5,000, H3K9ac (Abcam ab4441)‐1:1,000, H3K18ac (Abcam ab1191)‐1:1,000, β‐tubulin (Abcam ab6046)‐1:10,000, SIRT6 (Cell Signaling #12486)‐1:1,000, Lamin A/C‐1:1,000 (Abcam ab108595, Millipore 05–714), γH2AX (Millipore 05–636), PARP1(Abcam ab227244)‐1:1,000, Vimentin‐1:1,000 (Abcam ab92547); TAF15‐1:2,000 (Abcam ab134916); mADPr 1:500 (AbD33204 and AbD33205)(Bonfiglio et al, 2020 (link)).