Figure 5

Immune response to LL37 requires clathrin-dependent endocytosis (a) IL-6 mRNA in NHEKs cultured with various endocytosis inhibitors (Monodansylcadaverine (MDC): 200 μM, Pitstop-2^TM 25 μM, dynasore: 80 μM) for 30 minutes, treated with LL37 (2.5 μM) and U1 RNA (2.5 μg/mL) for a further 6 hours. (n = 3). (b) IL-6 mRNA in PMA-treated THP1 after treatment with cathelicidin peptides (3 μM) for 10 minutes, then stimulated with U1 RNA (12.5 μg/mL) overnight. (n = 3). (c) Gene sets induced in NHEK by LL37 + U1 RNA (fold change >2 versus vehicle control) and genes of identically treated cells first repressed by Pitstop-2^TM (25 μM) (fold change < −1.5). (d) Gene Ontology analysis gene set in c that was both induced by LL37 + U1 RNA and also inhibited by Pitstop-2^TM. The number of genes related to each biological process is indicated in parentheses. (e) Dynamin (DNM1) mRNA and clathrin (CLTC) mRNA measured in NHEKs after siRNA targeting each gene. (n = 3). (f) IL-6 mRNA in NHEKs following siRNA targeting of DNM1 or CLTC, and addition of LL37 (2.5 μM) and U1 RNA (2.5 μg/mL) for 6 hours. (n = 3). Data presented are from one representative experiment of at least two independent experiments. Error bars are SEM of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-hoc test. See also Fig. S5.