Figure 2. CCL2 promotes prostate cancer cell migration and induces CCL22 secretion, which is a ligand of CCR4.

A. Prostate cancer cells are placed in transwell inserts and treated with CCL2 (0–30 ng/ml). After 24-h incubation, PC-3 and DU145 cells that had migrated through the membrane are stained. The mean OD value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Data are presented as mean ± SD. B. DU145 cells are co-cultured with THP-1 and U937 cells and treated with CCL2 (30 ng/ml) for 24 h, CM is collected, and CCL17 and CCL22 levels are analyzed using ELISA. The mean OD value is read using a microreader at 450 nm, and data are presented as mean ± SD. C, D. Total RNA and protein are extracted from prostate cancer cells, and CCR2 and CCR4 gene and protein expression levels are analyzed using PCR (C) and western blot (D). E. Prostate cancer cells (1.0 × 10⁵ cells/well) are seeded into 6-well plates and cultured until they reach 60%–70% confluence. Cells are incubated with anti-CCR2 or anti-CCR4 antibody and detected using a second antibody conjugated with FITC (green). Cells are counterstained with 4',6-diamidino-2-phenylindole (blue). Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. All experiments are performed in triplicate, and mean values are shown. *p < 0.05, **p < 0.01, ***p < 0.001.