For ChIP, a 9-cm dish of subconfluent HaCaT cells (10-15 × 106 cells) was fixed with 1% formaldehyde for 10 min at room temperature. The formaldehyde was neutralized by the addition of 125 mM glycine for 5 min. The cells were washed twice in ice-cold PBS and collected by scraping in 1 mL 1% sodium dodecyl sulfate (SDS), 100 mg/L sonicated salmon sperm DNA, and protease inhibitors (15 mg/L aprotinin, 2 mg/L leupeptin, 0.2 g/L phenylmethanesulfonyl fluoride [PMSF]). To eliminate free FOXO1, the lysates were vortexed and insoluble material was collected by centrifugation at 14,000 ×g at 4°C for 5 min. The pellets were resuspended in 1 mL 0.25% SDS/200 mM NaCl/100 mg/L sonicated salmon sperm DNA and protease inhibitors and were sonicated to an average fragment size of 1 kb using a microtip on a sonicator (Branson 8150, USA). The remaining insoluble material was removed by centrifugation at 14,000 ×g at 4°C for 5 min. The supernatant was diluted with 2 vol 1% Nonidet P-40/350 mM NaCl and incubated for 12 h at 4°C with 10 μL antibody-coated paramagnetic protein G beads (Dynal, Inc. New York, USA)). Each ChIP used 1 μg DO-7 (anti-p53) and 1 μg PEP 2 (anti-Sp1) (Santa Cruz Biotechnology). Immune complexes were collected with a magnet, washed four times in 1% Nonidet P-40/350 mM NaCl/100 mg/L sonicated salmon sperm DNA, resuspended in 125 μL 1% SDS/16 mg/L salmon sperm DNA, and eluted by heating to 85°C for 10 min. Crosslinking was reversed by incubating the eluate for 6 h at 65°C. Samples were diluted with 125 μL water containing 160 mg/L proteinase K, and incubated for 1 h at 50°C. DNA was purified by phenol/chloroform extraction and precipitated with isopropyl alcohol and glycogen.

qPCR was performed on a PE5700 PCR machine using a TaqMan Master Mix (t (Applied Biosystems, USA).). The PCR cycles were 50°C for 2 min to digest dUTP-containing DNA, 95°C for 10 min to activate the Taq Gold polymerase, and then 40 cycles of 95°C for 15 s and 60°C for 1 min, except for the GAPDH primers, for which the annealing temperature was 55°C. Total RNA was extracted using TRIzol (Invitrogen), and 2 μg RNA were reverse-transcribed using SuperScript II (Invitrogen). The probe and primer sequences are showed in supplementary table 3.

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