Hbd 1

In order to know which defensin more efficiently inhibited E. coli proliferation, we incubated a constant load of bacteria (1 × 10⁵ CFU/mL) in the presence of 2000 ng/mL of each recombinant, HBD-1, HBD-2, HBD-3, or HBD-4, alone (Peprotech, Minneapolis, MN, USA. 300-51A, 300-49, 300-52, and 300-65, respectively) or combined (500 ng HBD-1 + 500 ng HBD-2 + 500 ng HBD-3 + 500 ng HBD-4). The experimental microbicidal assay was developed in DMEM antibiotic-free medium. As a control for bactericidal activity, a commercial ThermoFisher (Waltham, MA USA) antibiotic mixture was used (15240062): penicillin 100 U/mL, streptomycin 100 µg/mL, and amphotericin B 25 ng/mL. The bacteria were maintained at 37 °C in a humidified chamber with 5% CO₂. The viable bacteria were estimated by CFU counts in TSA plates from each experimental treatment at time 0 h and 4 h. Each treatment was repeated on at least four different occasions in triplicate.

Cytospin slides were made from PBAL and stained with May-Grünwald-Giemsa. A minimum of 300 cells were counted for differential cell counts by an observer blinded for the patient’s data. The AMPs, SLPI [21 (link)], and hBD-1 (PeproTech, London; UK) and hBD-2 (Antigenix America, Melville, NY, USA), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA), performed at the Leiden University Medical Center, the Netherlands.

HSC-3, UM1, SCC-9 and SCC-25 cells stably transfected with pGChBD-1 or pGCcontrol plasmids were seeded in 96-well plate at the concentration of 1×10³ cells/well. Every 24 hours after seeding, relative number of living cell was assessed using Cell Counting Kit-8 (CCK-8, Dojindo). Meanwhile, HSC-3, UM1, SCC-9 and SCC-25 cells were treated with 50 µg/ml recombinant hBD-1 (Peprotech), and cell viability with or without hBD-1 treatment was also assessed by CCK-8 assay.
For colony formation assay, cells were seeded in 6-well plate at the concentration of 3×10² cells/well. 14 days after seeding, cells were fixed with 4% paraformldehyde and then stained with crystal violet. Colony formation rate = colony number/cells seeded×100%.
The effect of exogenous expression of hBD-1 on apoptosis of HSC-3, UM1, SCC-9 and SCC-25 cells was assessed by Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining. Briefly, 48 hours after transfection with pGChBD-1 or pGCcontrol plasmids, 2×10⁵ trypsinezed cells from each group were collected and stained according to the instructions of the Annexin V-FITC Apoptosis Detection Kit (R&D Systems) and analyzed by a flow cytometer (Beckman Coulter). The data was presented as dot plots showing fluorescence intensity of Annexin-V FITC and PI. The percentage of apoptotic cells (Annexin-V+/PI− and Annexin-V+/PI+) was calculated.