The PFU were counted with ELISPOT reader (Ziess, Germany). Percentage of H5N1 inhibition was then calculated. Cell death reflecting cytopathic effect of H5N1 infection was observed under a microscope. All experiments with H5N1 virus were performed in a Biosafety Level 3 facility. MxA siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Briefly, semi-confluent HGECs were seeded with growth media without antibiotics 1 day before transfection. 80 nM MxA siRNA and 5 μL siRNA tranfection

reagent were diluted in 1 mL of transfection medium, mixed, and incubated at room temperature for 45 min. HGECs were washed two times with transfection medium and then the dilutes MxA siRNA was added for 7 h, then 2× growth medium was added and PLX4032 purchase cells were cultured overnight. Depletion of MxA expression by MxA siRNA was assessed by real-time RT-PCR and immunohistochemistry (for protein level). Transfected HGECs were treated with α-defensin-1 overnight and then infected with H5N1 virus. Highly purified PMNs from healthy human subjects were prepared by density centrifugation

using Polymorphprep™. The purity of PMNs was > 95%, as determined by anti-CD16 mAb using flow cytometry. PMNs (5×106 cells/mL) were incubated for 6 h in serum-free keratinocyte growth medium. Supernatants were collected for measurement of α-defensin production by ELISA (detected all human α-defensin-1, -2, and -3). PMN supernatants with or without neutralizing antibody against α-defensins (neutralizes all human α-defensin-1, -2, and -3; 1 μg/mL) or neutralizing antibodies against IFN-α (400 neutralization PXD101 chemical structure unit/mL) and IFN-β (400 neutralization unit/mL) was added to HGEC cultures. After 6 h of treatment with either PMN supernatant or medium control, mRNA expression of MxA was analyzed by real-time RT-PCR. After 24 h incubation, MxA protein expression in HGECs was analyzed by immunohistochemistry. The parametric Student’s t-test was used for normally distributed data, and the nonparametric Mann–Whitney rank-sum test was used for nonnormally

distributed Tideglusib data. A p-value < 0.05 was considered statistically significant. Data were analyzed with SPSS Version 11.5 software (SPSS Inc., Chicago, IL, USA). This work was supported by BRG5380011 from Thailand Research Fund, Chulalongkorn University, and Ratchadapisek endowment. The authors thank S. Wiboon-ut (Department of Microbiology, Faculty of Science, Mahidol University) for technical assistance with avian influenza H5N1 experiments. We also thank Dr. C. Champaiboon for tissue sample collection, P. Ekchariyawat for STAT1 activation experiment, and Dr. K. Torrungruang for valuable comments and suggestions. Conflict of interest: The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.