Chromatin immunoprecipitation (ChIP)

The hADSCs were infected with or without Ad2 or Ad36 for 48 h and 72 h, respectively. Magna ChIP G Chromatin Immunoprecipitation Kit (Millipore Corporation, Billerica, MA, USA) was used to identify the regions of the genome associated with FoxO1 or PPARγ. The CHIP assays were performed as previously described, with modifications [17] Briefly, cells were incubated for 10 min in 1% formaldehyde and then quenched with glycin for 5 min. After washing and cellular and nuclear lysis, chromatin breakdown was performed with an Ultrasonic Cell Disruptor (LANYI Shanghai, China). A preclear step was performed (1 h at 4 °C with 40 μl of magnetic beads) before overnight incubation of chromatin and beads with anti-FoxO1 or anti- PPARγ antibodies (CST, Danvers MA, USA). Cells were also submitted to a no-antibody condition (mock condition) to exclude non-specific binding. DNA purification was performed using the silica columns provided with the kit (Magna ChIP G, Millipore). The purified DNA samples were used as templates for qPCR detection. The primers used were shown in Table 1.

The primers of CHip-PCR

The following PCR conditions were used: denaturation at 94 °C for 10 min, followed by 35 cycles of 94 °C for 20 s and 60 °C for 1 min. Each sample was repeated 3 times. The bands were quantified by relative quantitative analysis and calibrated with the amount of input.