RNA extraction, reverse transcription PCR (RT-PCR) and ChIP-PCR analysis
Cells were collected and lysed in Trizol (Takara, Tokyo, Japan). RNA samples were used for reverse transcription with Oligo (dT) primers (Takara, Tokyo, Japan). The cDNA products were subjected to RT-PCR. cDNA amplification was performed for 35 cycles (94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s) using Tag DNA polymerase (Promega, Shanghai, China). The RT-PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The Gel Imaging and Documentation DigiDoc-It System (version 1.1.23; UVP, Inc., Upland, CA) was used to scan the gels. Gapdh was performed as a loading control. The primer sequences used in this study were as follows:
IGF-1: forward, 5′-GCTCTTCAGTTCGTGTGTGGA-3′;
reverse, 5′-GCCTCCTTAGATCACAGCTCC-3′;
PCNA: forward, 5′-CCTGCTGGGATATTAGCTCCA-3′;
reverse, 5′-CAGCGGTAGGTGTCGAAGC-3′;
PCNA: forward, 5′-AGGCACTCAAGGACCTCATCA-3′;
reverse, 5′-GAGTCCATGCTCTGCAGGTTT-3′;
β-actin: forward, 5′-CATGTACGTTGCTATCCAGGC-3′;
reverse, 5′-CTCCTTAATGTCACGCACGAT-3′.
For ChiP primers, they were as follows:
IGF-1:forward, 5′-CAGGTCTGGCTCATTTCCATC-3′; reverse, 5′-GCGCTTTCCATGGCTGTC-3′; probe, 6FAM-CCCCTGGGAAAGCACACCTGGA; PCNA: forward, 5′-CCACCATAAAGCTGGGGCTT-3′; reverse, 5′-TCTCCCCGCCTCTTTGACTC-3′.
probe, 6FAM-CCCCTGGGAAAGCACACCTGGA