Regulation of CCL2 Gene Expression Figure 1. CCL2 mRNA accumulation is induced by pro-inflammatory cytokines in rat islets and b-cell lines. A. 832/13 rat insulinoma cells were treated with 0.1, 1, and 10 ng/mL of IL-1b for 6 h. B. 832/13 cells were stimulated with 1 ng/mL IL-1b for 0, 3, 6 and 12 h. C. Rat islets were either untreated or treated with 10 ng/mL IL-1b for 6 h. A–C. Total RNA was isolated and CCL2 mRNA levels were measured and normalized to those of the housekeeping gene, Ribosomal S9 (RS9). *p,0.05 vs. NT. D. 832/13 cells were transfected with 3.6 kb of the CCL2 promoter upstream of the transcriptional start site fused to a luciferase reporter (3.6-Luc). 24 h post-transfection cells were stimulated for 4 h with 1 ng/mL IL-1b. Relative promoter activity of 3.6-Luc was measured and normalized to protein content via BCA assay. #p,0.001 vs. NT. E. 832/13 cells were untreated or treated with 1 ng/mL IL-1b for 0, 3, 6 and 12 h. CCL2 release into the media was measured by ELISA and was normalized to protein content via BCA assay. #p,0.001 vs. 0 h, **p,0.01 vs. 0 h. Data are means 6 SEM from 3–4 individual experiments. doi:10.1371/journal.pone.0046986.g001 complex (Figure 2G, bottom). Moreover, the particular NF-kB complex associated with the CCL2 genomic response element was impaired in the presence of antisera targeted against either p65 or the coactivator p300, but not with antisera against p50 (Figure 2G). The DNA binding data is thus consistent with NF-kB protein requirements for activation of the CCL2 gene (see Figures 2 E–G). PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46986