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. 2021 May 26;297(1):100828. doi: 10.1016/j.jbc.2021.100828

Figure 1.

Figure 1

Sequential alanine substitution in LL-34 reveals amino acid residues critical for innate vetting activity in vitro.A, normal human keratinocytes (NHEKs) were treated with either U1 dsRNA (2.5 μg/ml) or a combination of U1 dsRNA (2.5 μg/ml) and LL-34 peptides (LL-34 L1A and R34A and LL-34 parent) at 2 μM for 18 h. Protein levels of human IL-6 were analyzed from cell supernatant with ELISA (n = 3). Corresponding MIC values of LL-34 peptides L1A–R34A against Staphylococcus aureus USA300 are shown in the heat map. B, human dermal endothelial cells (HDMECs) were treated with either U1 dsRNA (2.5 μg/ml) or a combination of U1 dsRNA (2.5 μg/ml) and LL-34 peptides (Pep 1–Pep 34 and Pep–parent LL-34) at 2 μM for 18 h. IL-6 ELISA results are shown (n = 3). Green bars denote loss of IL-6 protein secretion compared with parent control shown in red. C, helical wheel plot for LL-34. Amino acids with green circles denote specific residues where replacement with an alanine caused the loss of IL-6 secretion compared with parent LL-34 peptide in NHEKs cotreated with U1 dsRNA. D, in the helical wheel plot for LL-34, circled green amino acids indicate that a substitution caused a loss of vetting in HDMEC. E–H, NHEKs were treated with LL-34-parent peptide (LL-34-P), LL-34-F5A, LL-34-I24A, LL-34-F27A, and LL-34-L31A at 2 μM, either individually or in combination with U1 dsRNA (2.5 μg/ml) for 8 h. Gene expression of IL-6, IFNB1, TSLP, and CXCL10 was assessed with qRT-PCR. GAPDH was used as a reference gene (n = 3) (∗p < 0.05; ∗∗∗∗p < 0.0001). One way-ANOVA was used. IL-6, interleukin 6; LL-34, 34-amino acid peptide; MIC, minimum inhibitory concentration, ns, not significant.