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. 2023 May 30;18(5):e0285597. doi: 10.1371/journal.pone.0285597

Fig 3. shRNA-mediated ACKR3 knockdown does not diminish AM scavenging in TNFα/IFNγ stimulated primary adLECs or jdLECs.

Fig 3

adLECs and jdLECs were transduced with ACKR3-specific shRNA constructs (A: no knockdown, B,C; 50–80% knockdown–see S3 Fig), scrambled shRNA (shRNA Ctrl) or untransduced control LECs (Ctrl). Cells were stimulated by overnight incubation with TNFα/IFNγ. Subsequently, cells were incubated with either CXCL11/12-AF647 (50nM) or AM-AF568 (50nM) and uptake analysed by flow cytometry. While CXCL11/12-AF647 scavenging correlated with ACKR3 knockdown in (A) adLECs and (C) jdLECs, no impact of ACKR3 levels on AM-AF568 scavenging was observed in (B) adLECs and (D) jdLECs. (E) Also when performing the experiment with 500nM AM-AF568, no impact of ACKR3-knockdown on scavenging was observed. Data from three or four independent experiments are shown as mean ±SD. Each data point represents one replicate. Two-way ANOVA, followed by Tukey’s multiple comparison test (A, C). Mixed effects model, followed by Tukey’s multiple comparison test, was applied, when repeated measures pairing was incomplete, due to subclone availability (B, D, E). A p-value of p≥0.05 was considered not significant (ns).