Figure 5

c-FOXP3 directly activates CCL5 expression. (a) The mRNA level of CCL2, CCL3, CCL4, CCL5, CCL17, CCL20, CCL22 and CCL28 were measured by real-time–PCR (mean±s.e.m., n=3; *P<0.05 compared with Control, by Student’s t-test). (b) The protein level of c-FOXP3 and CCL5 in Panc-1 and Pan02 were detected by western blotting. (c) The secretion of CCL5 in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA; mean±s.e.m., n=3; *P<0.05 compared with Control, by Student’s t-test). (d) c-FOXP3 and CCL5 expression in both normal pancreas and PDAC by immunohistochemistry. Magnification: × 200. (e) Specificity of the ChIP assay. Human CCL5 gene, including the FOXP3-binding motif 1–4 (upper). Binding of FOXP3 and CCL5 promoter was observed at motif-4. p21 was used as positive control (down). (f) The Panc-1 cells were transfected with either vector control or FOXP3 in conjunction with the luciferase reporter pGL3-CCL5-promoter (motif-4 WT) or pGL3-CCL5-promoter (motif-4MUT) vectors. pGL3-p21-promoter was used as positive control. After 48 hours, firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega, Madison, WI, USA) and the ratio was determined (*P<0.05, by Student’s t-test). The experiment was performed in triplicate and repeated three times with the same results.