Human peripheral blood cd14 monocytes

All media described below were supplemented with 10% fetal bovine serum (Sigma-Aldrich). Human airway epithelial A549 cells were maintained in F-12 K media (Gibco), BEAS-2B cells in RPMI 1640 media (Gibco). BEAS-2B cells stably expressing human IRAK-M were obtained by plasmid transfection following geneticin selection (300 μg ml⁻¹). Human primary bronchial epithelial (Lonza) cells were maintained in BEGM (bronchial epithelial growth media) supplemented with BEGM SingleQuots. Human peripheral blood CD14+ monocytes (Lonza) were cultured in RPMI 1640 media (Gibco) containing 1 mM pyruvate and GM-CSF (50 ng ml⁻¹; R&D systems). MEF cells were obtained from E13 embryos and maintained in DMEM (Corning Cellgro). p65^−/− MEFs reconstituted with p65 WT were cultured in DMEM (Corning Cellgro) containing puromycin (1.5 μg ml⁻¹). All cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C.

Total RNA was isolated using the Nucleospin^® RNA kit protocol (Macherey–Nagel, Germany). Reverse transcription of total RNA was performed using the Prime Script RT kit (Takara, Takara Bio, Japan); qRT-PCR was performed with the ABI PRISM^® 7500 sequence detection system (Applied Biosystems, USA) using SYBR Premix Ex Taq™ II (Tli RNaseH plus, Takara Bio, Japan) and gene-specific primers for SPI1 and GAPDH (Table 1). PCR cycling conditions consisted of initial denaturation at 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Data was analyzed using the 2^−ΔΔCt method [25 (link)]. Human peripheral blood CD14⁺ monocytes (Lonza, Basel, Switzerland) were kept as a positive control for the comparison of SPI1 expression.