Fig. 2

Aspirin promoted OLA1P2 transcription through FOXD3 upregulation. a The biotin-labeled OLA1P2 promoter was mixed with the nuclear extract separated from DMSO/aspirin-treated primary culture cancer cells. The eluted proteins were then analyzed using mass spectrometry methods. b The protein levels in the nuclei of primary cultured cells treated with aspirin (100 μM) for 48 h were analyzed using immunoblotting methods. c Protein levels in primary culture cancer cells transfected with shRNA vectors for 48 h were analyzed by immunoblot. d OLA1P2 expression was analyzed by qRT-PCR methods in primary culture cancer cells transfected with the shRNA vector for 48 h and then treated with aspirin (100 μM) for 48 h. e QRT-PCR analysis of FOXD3 mRNA expression in CRC cells treated with aspirin (top lanes). Immunoblot analysis of FOXD3 protein levels in CRC cells treated with aspirin (middle lanes). Quantitative methylation-specific PCR analysis of FOXD3 promoter methylation levels in CRC cells treated with aspirin (bottom lanes). M: methylation-specific primer; U: unmethylation-specific primer. f Diagram showing conserved FOXD3 response elements in the OLA1P2 promoter. g ChIP assays using anti-FOXD3 or anti-IgG antibodies were performed to determine the affinity of FOXD3 for the OLA1P2 promoter in primary culture cancer cells. h Primary culture cancer cells were co-transfected with the lenti-FOXD3 vector and a luciferase reporter for 48 h. i Primary culture cancer cells were transfected with a luciferase reporter for 12 h and were then treated with aspirin (100 μM) for 48 h. **: P <0.01; ***: P <0.001