Fig 2. AM scavenging in primary ndLECs is not reduced by pharmacologic manipulation of ACKR3.

(A) Structure of the custom-synthetized, disulfide bridged AM-AF568 according to [31]. The unnatural amino acid L-propargyl glycine (Pra) was inserted at position 9 of the AM sequence to allow for site-specific fluorescent labeling (see Materials and methods). (B) AM-AF568 induces comparable LEC proliferation as unlabeled AM. Each data point represents the mean of one experiment with 10 replicates. Mixed-effects analysis, Šídák’s multiple comparison test. (C, D) Uptake of either 50nM AM-AF568 or 50nM CXCL11/12-AF568 by unstimulated or TNFα/IFNγ stimulated LECs in presence or absence of CCX771 was quantified by FACS. Effect of CCX771 on uptake of (C) AM-AF568 and (D) CXCL11/12-AF647. Pooled data from 3 experiments are shown. One-way ANOVA, followed by Tukey’s multiple comparison test. (E, F) ndLECs were incubated with (E) AM-AF568 (500nM) or (F) CXCL11/12-AF647 (50nM) at either 4°C or 37°C and uptake into cells was analysed by confocal microscopy after 1h. Representative images from two independent experiments. Scale bars: 15μm. (G-I) LECs were incubated with either 50nM, 500nM or 1μM AM-AF568 in presence or absence of CCX771, and uptake was analysed by flow cytometry. (G) Representative histogram showing AM-AF568 internalization in stimulated LECs incubated with 50nM and 500nM. (H, I) Pooled data from three independent experiments performed with (H) unstimulated LECs and from two independent experiments performed with (I) TNFα/IFNγ stimulated LECs are shown. Results are shown as mean ±SD. Two-way ANOVA, followed by Tukey’s multiple comparison test. A p-value of p≥0.05 was considered not significant (ns).