CCR8 expression and function in tumor cell lines. (A) Expression of CCR8 mRNA (RT-PCR, in vitro) and protein (Western blot, in vitro and in vivo) by MDA-MB-435 and SK-MEL-25 cells. (B) FACS analysis was performed on cultured tumor cells and primary melanocytes. (C) Effects of CCR8 antagonist MC148 on tumor cell chemotaxis to LEC-CM. Error bars indicate mean ± SD, n = 3. *, P < 0.05. (D) Effects of CCR8 antagonist MC148 and pertussis toxin (PTx) on cytoskeletal rearrangements induced by rhCCL1 in MDA-MB-435 cells. Cells are stained with phalloidin (red) and Hoechst (nuclei, blue). Data are representative of three experiments. (E and F) Changes of cytosolic Ca²⁺ concentration measured by FACS in Fluo-4–labeled cells upon addition of human (E) or mouse (F) CCL1 (12.5 or 25 nM as indicated). The black arrow indicates the time of CCL1 addition. MC148 was added at 25 nM. Data are expressed as percentile fluorescence intensity of Fluo-4 dye bound to Ca²⁺. Data are representative of at least two experiments. (G) Cell shape change of MDA-MB-435 cells stimulated with 50 ng/ml mouse CCL1 for the times indicated. Cells are stained with rhodamine-labeled phalloidin (red). Bars, 10 µm.