Human beta-defensin-3 activates professional antigen-presenting cells via Toll-like receptors 1 and 2 -- Funderburg et al. 104 (47): 18631 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Funderburg et al. 10.1073/pnas.0702130104.

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SI Figure 5
SI Figure 6
SI Figure 7

SI Figure 5

Fig. 5. hBD-3 does not stimulate HEK293 cell lines expressing single TLRs. HEK293 cells expressing the indicated TLR were cultured in medium alone, or medium supplemented with shBD-3 (20 mg/ml) or with positive controls for each of the TLRs [heat-killed Listeria monocytogenes (HKLM), 10⁸ cells per milliliter; poly(I:C), 1 mg/ml; LPS, 100 ng/ml; flagellin, 1 mg/ml; Loxorobrine, 1 mM; ssRNA 40, 5 mg/ml; CPG ODN 2006, 1 mg/ml]. NF-kB activation was monitored by secretory alkaline phosphatase reporter gene expression. hBD-3 did activate reporter gene expression in any of the TLR-expressing cell lines. Experiments were performed in duplicate.

SI Figure 6

Fig. 6. hBD-3 does not activate mouse bone marrow-derived macrophages. Macrophages were derived from femur marrow precursors by 7-day culture in DMEM/10% FBS (D10F) medium supplemented with 20% LADMAC conditioned medium. Macrophages were subsequently incubated overnight in D10F. The medium was replaced with fresh D10F or D10F supplemented with various concentrations of shBD-3 or Pam₃CSK₄ ranging from 0 to 5,000 nM. After centrifugation of the cell culture plates, supernatants were collected and TNF-a was measured by ELISA (BD Biosciences). hBD-3 did not stimulate TNF-a production from the WT or TLR2^-/- macrophages. Pam₃CSK₄ induced TNF-a production from WT macrophages but not from TLR2^-/- macrophages.

SI Figure 7

Fig. 7. Disruption of the structure of shBD-3 results in loss of activity. shBD-3, or Pam₃CSK₄, was heated to 95°C for 30 min to disrupt secondary and tertiary structure. Whole PBMCs were isolated from healthy control donors (10⁶ cells per 500 ml) and incubated in medium alone, or in medium supplemented with 20 mg/ml shBD-2, shBD-3, boiled shBD-3, or 100 ng/ml Pam₃CSK₄ or boiled Pam₃CSK_4. Synthetic hBD-2 failed to induce heightened levels of costimulatory molecules (results are representative of 10 experiments). Boiling shBD-3 resulted in ≈90% reduction in its ability to induce CD80. The activity of Pam₃CSK₄ was not affected by boiling (results are representative of three experiments).