ChIP Assay for FoxO1 Binding

ChIP assay was performed in cultured HepG2 and HEK-293 cells, either untreated or pretreated with siRNA targeting HMGA1, as described previously^{23 (link),70 (link)}. For in vivo ChIP, at the end of the indicated treatments, mice were killed by cervical dislocation, the liver was rapidly removed, prewarmed with PBS, and treated with 0.2% collagenase for 15 min. The liver was then diced, forced through a 60 µm stainless steel sieve, the hepatocytes were collected directly into DMEM containing 1% formaldehyde, and the formaldehyde-fixed DNA-protein complexes were immunoprecipitated with the anti-FoxO1 antibody sc-11350 from Santa Cruz Biotechnology. Sequence-specific primers for the IGFBP1 gene promoter used for PCR amplification of ChIP-ed DNA (30 cycles), using PCR ready-to-go beads (Amersham Pharmacia Biotech): human IGFBP1 (NT_007819) for 5′-CAGAAAGAGAAGCAATTCCG-3′, rev 5′-TACCAGCCAGACGCGAGCAA-3′; mouse Igfbp1 (NT_039515) for 59-CCTGGGGAGGGAGAAACAACT-39, rev 59-GCAGTGTTCAATGCTCGCTGG-39. PCR products were electrophoretically resolved on 1.5% agarose gel and visualized by ethidium bromide staining.