Figure 3.

PGE₂ promotes CXCL1 expression and release. (A) Human antibody array analysis was used to determine the differences in the release of human angiogenic factors from the conditioned medium of LS-174T cells treated with vehicle (left) or PGE₂ (right). The positive controls produced by biotin-conjugated IgG can be used to compare the relative expression levels among the different membranes. (B) Quantitative real-time PCR analysis of the mRNA level of CXCL1 in four CRC cell lines and one breast cancer cell line. The cells were incubated in serum-free media for 24 h and treated with 1 μM PGE₂ for 4 h. The relative expression of target gene is averages of triplicates that are normalized against the transcript levels of hActin. Data are represented as the mean ± SE of the relative expression from three independent experiments. Asterisks represent statistical differences (P < 0.05; Student's t test). (C) PGE₂ stimulates CXCL1 promoter activity. The LS-174T cells were transiently transfected with CXCL1 (−306 to +45) luciferase reporter gene and pRL-SV40 plasmids followed by treatment with PGE₂ for 24 h. The dual luciferase assays were performed as described in Materials and methods. Data are represented as the mean ± SE of relative luciferase activity from three independent experiments. (D) The levels of CXCL1 protein in cell supernatants were determined by ELISA. Bar graphs show the values of CXCL1 concentration in the conditioned medium of LS-174T cells treated with indicated amounts of PGE₂ for 24 h, adjusted for cell number. Three independent experiments in duplicate were performed.