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. 2017 Oct 6;8:1233. doi: 10.3389/fimmu.2017.01233

Figure 5.

Figure 5

Chemerin, but not CCL19, is internalized by C-C chemokine receptor-like 2 (CCRL2)-transfected HeLa cells. (A) Hela cells stably expressing CCRL2 (CCRL2) or control cells (CTR) were incubated with 1 nM chemerin or 1 nM CCL19. At the indicated times, supernatants were collected and ligand concentration was assessed by enzyme-linked immunosorbent assay (ELISA). The mean values of three independent experiments are shown; the values for each ligand were normalized over the value of the respective control at 1 h of incubation. ***p < 0.001 (CCRL2 + chemerin vs. CTR + chemerin) by two-way ANOVA. (B) Hela cells stably expressing CCRL2 (CCRL2) or control cells (CTR) were pre-incubated with 80 µM Dynasore or 0.1% DMSO for 1 h, then chemerin was added to the final concentration of 1 nM. After 1 h incubation, supernatants were collected and chemerin concentration was assessed by ELISA. Chemerin concentration was normalized over the one of DMSO-treated control cells. The mean values of three independent experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.001, by one-way ANOVA followed by Bonferroni’s multiple comparison test. (C) Hela cells stably expressing CCRL2 (CCRL2) or control cells (CTR) were incubated for 2 h in the absence (no chemerin) or in the presence of 1 nM chemerin. Cells were then washed with PBS (PBS), acidic buffer (pH3) or with acidic buffer followed by high salt buffer (pH3 + NaCl), for 5 min. Then, cells were lysed and whole cell lysates were analyzed by ELISA for chemerin concentration. The mean values of three independent experiments are shown. ***p < 0.001, by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Chemokine-like receptor 1 (CMKLR1)- or CCRL2-expessing HeLa cells or control cells were incubated with 1 nM chemerin for the indicated times. Supernatants were collected and chemerin concentration was assessed by ELISA. The values were normalized over no cells, at 1 h of incubation; the mean values of three independent experiments are shown. *p < 0.05 (CMKLR1- vs. CCRL2-expressing cells) by two-way ANOVA; CTR, control HeLa cells; CCRL2, HeLa cells stably expressing mCCRL2; CMKLR1, HeLa cells stably expressing mCMKLR1; no cells, supernatant incubated in the absence of cells; Dyn, Dynasore; ns, not significant.