Inflammatory cytokines increase CCL1 production by LECs and tumor cell migration to LEC-CM. (A) Real-time qPCR analysis for CCL1 mRNA upon treatment of LECs with 50 ng/ml TNF, 500 ng/ml LPS, or 50 ng/ml IL-1β for 3, 6, and 18 h. Error bars indicate mean ± SD, n = 3. (B) RT-PCR for CCL1 mRNA expression in cultured LECs. cDNA from PMA-stimulated Jurkat cells (50 ng/ml, 48 h) was used as a positive control. (C and D) Western blot (C) and ELISA (D) for CCL1 protein in conditioned medium from LECs treated with 100 ng/ml TNF, 500 ng/ml LPS, or 50 ng/ml IL-1β for 48 h, and CM from Jurkat cells treated with 50 ng/ml PMA for 48 h. Error bars indicate mean ± SD, n = 2. (E and F) Effects of CCL1 depletion on tumor cell (MDA-MB-435) migration to LEC-CM generated in presence of 50 ng/ml TNF (E) or 500 ng/ml LPS (F) for 48 h. Bars indicate mean ± SD, n = 3. Data are representative of three experiments. *, P < 0.05. (G–L) Immunostaining of mouse skin for either LYVE-1 or podoplanin (both green) and for CCL1 (red); boxed areas in J and L depict the overlay. Images are representative of at least six skin samples per group. Arrows, lymphatic vessels; ep, epidermis. Bars, 50 µm.