Cells were resuspended with PBS and sonicated by adding ChIP nuclear lysis buffer to obtain the ideal chromatin fragments with a length of 150–1000 bp for IP reactions. The fragments were treated overnight with magnetic beads and antibody (anti-RNA Pol II, 5:25, ab26721, Abcam) followed by de-crosslinking. Purified DNA was obtained using a DNA purification kit (TIANGEN, DP214-02, Biolink Biotechnology, Beijing, China). Subsequently, quantitative polymerase-chain reaction (PCR) was performed to assess the enrichment level of RNA Pol II at the binding sites of the VEGF promoter, with total chromatin as standard changes (input). Rabbit IgG antibody (ab172730, Abcam) was used as the negative reference, and primers for the VEGF promoter region were designed: VEGF-F 5’-3’: GACGTTCCTTAGTGCTGGCGGGTAGGTTTGA; VEGF-R 5’-3’: GGCACCAAGTTTGTGGAGCTGAGAAC. Reaction conditions were as follows: pre-denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 10s, annealing at 55°C for 30s, and extension at 62°C for 20s. The PCR products were subjected to electrophoresis on 1% Tris-acetate-ethylenediamine tetraacetic acid agarose gels in triplicate [53 (link),54 (link)].