Immunofluorescence 832/13 cells were seeded onto glass cover slips within a 6-well plate. Following treatments described in the Figure Legends, the cells were fixed using 4% paraformaldehyde for 20 min, followed by PBS washing three times, five minutes each time. After the wash steps, glass cover slips were moved to a humidified chamber and exposed to a blocking solution containing 10% Goat Serum (Sigma, St. Louis, MO), 0.25% Triton X-100, and 1X PBS for 1 h at room temperature. After this blocking incubation, the blocking solution was removed by aspiration and the primary antibody [p65 (Santa Cruz Catalog Number sc-372)] diluted in 10% Goat Serum, 0.25% Triton X-100, PBS was applied and left overnight at 4uC. After approximately 16 h of incubation with primary antibody, the cells were again washed with PBS three times for five minutes each time. After washing with PBS, cover slips were returned to the humidified chamber and incubated with secondary antibody (Alexa FluorH 488 goat anti-rabbit IgG (H+L) Invitrogen Molecular Probes A11008) for 1 h at room temperature. After the secondary antibody incubation, cover slips were washed with PBS – three times for five minutes each time. After washes were complete, cover slips were mounted onto glass slides using Prolong Gold w/DAPI Anti-fade reagent (Life Technologies Co.). These slides were analyzed by epifluorescence microscopy (Nikon Eclipse Ti-E) using a 60x objective lens (NA 1.49). 363 large image scans were collected and analyzed using NIS-Elements AR v3.1 software (Nikon Instruments, Melville, NY). Isolation of Nuclear Protein and Electrophoretic Mobility Shift Assays Subcellular fractionation and isolation of nuclear protein from 10 cm dishes was performed using the NE-PER kit from Thermo Scientific exactly as directed by the manufacturer’s protocol. Oligonucleotides corresponding to the NF-kB sequences present in the rat CCL2 gene promoter were synthesized with 59-biotin labels, purified by HPLC, and then annealed prior to incubation with 10 mg of nuclear protein. The sequence of the sense oligo (NF-kB sites underlined) is: 59-GGTCTGGGAACTTCCAATACTGCCTCA- GAATGGGAATTTCCACACT- 39. Cold competitor oligos (i.e., without biotin tags) were synthesized and purified in the same manner. The binding reactions were set up as described in the LightShift Chemiluminescent EMSA kit (Thermo Scientific). These reactions were then separated on 6% DNA retardation gels (Invitrogen), followed by transfer to nylon membranes. The protein-DNA complexes were crosslinked to the nylon membrane by using a UV transilluminator with 312 nm bulbs. Blocking of the membrane and subsequent downstream detection of the biotin label were exactly as described in the LightShift EMSA kit protocol. Statistical Analysis One way ANOVA analysis with Tukey’s post-hoc correction was performed with statistical significance at the 90%, 95% and 99% confidence intervals denoted in the figure legends. Results CCL2 mRNA Abundance is Increased by Pro- inflammatory Cytokines in Rat Islets and b-cell Lines CCL2 co-localizes with insulin-positive cells in the pancreatic islets [47,48]. In addition, the CCL2 gene promoter contains genomic elements, such as NF-kB and gamma activated sequences (GAS), which indicate potential responsiveness to pro-inflamma- tory cytokines. We therefore examined the effects of IL-1b and c- IFN, two distinct pro-inflammatory cytokines involved in b-cell Regulation of CCL2 Gene Expression death and dysfunction [49,50], on the expression of this gene using rat islets and b-cell lines. In 832/13 rat insulinoma cells, we observed that 1 ng/mL IL-1b is sufficient to drive maximal expression of the CCL2 gene, as increasing the amount 10-fold to 10 ng/mL does not promote additional mRNA accumulation (Figure 1A). Next, we examined CCL2 mRNA accumulation over time, noting that expression increased within 3 h, was maximal at 6 h, and then decreased by 12 h after exposure to 1 ng/mL IL-1b (Figure 1B). Expression of the CCL2 gene is also enhanced by IL- 1b in isolated rat islets (Figure 1C). We note that while there is a GAS response element present in the CCL2 gene promoter, c-IFN had no effect on the expression of the CCL2 gene in 832/13 cells while in rat islets, there was a small response (,20% of that produced by IL-1b; not shown). By contrast, transcription from 3.6 kb of the proximal CCL2 gene promoter linked to a luciferase reporter was strongly increased in response to IL-1b (Figure 1D). This transcriptional readout correlated with augmented secretion of CCL2 protein over time (Figure 1E), indicating that expression of the gene is most likely coupled to secretion of protein. While we observed low levels of CCL2 gene expression in the basal state (e.g., without IL-1b exposure), there was a marked induction in response to IL-1b (cycle threshold (Ct) values are given in Figure S1).Thus, we conclude that IL-1b drives the synthesis and secretion of CCL2 in rat pancreatic b-cells. The p65 Subunit of NF-kB is Both Necessary and Sufficient for Expression of the CCL2 Gene We next directly manipulated the p65 subunit of NF-kB to determine its involvement in the regulation of CCL2 gene expression by IL-1b. First, we used the IkBa super-repressor, which contains two amino acid substitutions (S32A/S36A) that eliminate phosphorylation-induced degradation of the regulatory protein in response to pro-inflammatory stimuli [41]. The NF-kB subunits are normally released from regulatory subunits, such as IkBa, for translocation to the nucleus after phosphorylation- induced regulatory subunit degradation [20]. Thus, the mutant IkBa protein attenuates NF-kB pathway actions by retaining p65 in the cytoplasm [41]. We observed that overexpression of the IkBa super-repressor blocked the IL-1b-mediated increases in CCL2 mRNA by 79%, promoter activity by 84%, and secretion by 68% (Figure 2 A–C). We then compared the wild-type CCL2 gene promoter (3.6– WT) to a construct that contains point mutations abolishing the known NF-kB binding sites (3.6–kBm) (Figure 2D). The NF-kB mutant reporter construct was refractory to stimulation by IL-1b, while the wild-type construct was robustly responsive (28-fold; Figure 2D). Since the wild-type CCL2 gene promoter is strongly induced by IL-1b, we further examined which NF-kB subunits were required for transcriptional induction in response to cytokines. Using siRNA-mediated suppression of p65 (76% decrease in mRNA - not shown) and corresponding decrease in both cytoplasmic and nuclear protein (Figure S2), we discovered that the CCL2 gene requires p65 for IL-1b-mediated transactiva- tion (Figures 2E–F). We next investigated binding to the NF-kB element present within the CCL2 proximal gene promoter using electrophoretic mobility shift assays. We discovered that NF-kB bound to this genomic region was detected only using nuclear fractions under IL-1b stimulated conditions (Figure 2G). Furthermore, the complex associated with the NF-kB response elements was abrogated by overexpressing the IkBa super-repressor construct (Figure 2G). To address specificity of the bound complex, we demonstrated that inclusion of fifty-fold molar excess (50 M X) of unlabeled competitor oligo diminished the specific protein-DNA PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46986