**2. Materials and Methods**

### *2.1. Cell Culture and Treatment of Primary Human Bronchial Fibroblasts*

The primary bronchial fibroblasts were isolated from endobronchial tissue biopsies obtained from patients with severe asthma or healthy volunteers. These fibroblasts were archived at Quebec Respiratory Health Research Network Tissue Bank (McGill University Health Centre (MUHC)/Meakins-Christie Laboratories Tissue Bank, Montreal, QC, Canada), as previously described [21]. The original study was approved by the MUHC Research Ethics Board (REB) with reference number 2003–1879 and the subjects had provided written informed consent in accordance with the Declaration of Helsinki. The fibroblasts were agematched with a mean age of 43.4 ± 8.3 years for the severe asthmatics and 43.7 ± 12.5 years for the non-asthmatics. Furthermore, cells were chosen from subjects who were non-smokers, as smoking patients with severe asthma demonstrated a distinct gene expression profile compared to non-smoking severe asthmatics and non-smoking individuals without asthma [22].

The cells were revived in Dulbecco's modified Eagle's medium (DMEM)—high glucose supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL of penicillin, and 100 ng/mL streptomycin in 75-cm2 flasks. The cells were maintained at 37 ◦C in 5% CO2 with medium change performed every 2–3 days. They were harvested when 80–90% confluent using 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and seeded into multiwell tissue culture plates for experiments.

The cells were seeded into 6- or 12-well plates at approximate cell density/well of <sup>10</sup> × 104, and 5 × 104, respectively. At ~70% confluency, they were serum-starved in FBS-free DMEM complete medium for a period of 24 h before experiments. For baseline measurements, the cells were cultured in DMEM complete medium thereafter. For LPS stimulation experiments, the cells were stimulated with 10 μg/mL of LPS (Sigma-Aldrich, Burlington, MA, USA, Cat. No. L8643) for the specified amount of time. Selective inhibition of Interleukin 1b Receptor-associated Kinase (IRAK) 1/4 was carried out to block the Bcl10 upstream signaling. Accordingly, the cells were pre-treated with 50 μM of IRAK 1/4 Inhibitor I (R&D Systems, Minneapolis, MN, USA, Cat. No. 5665/50) for 1 h prior to LPS stimulation for 2 h.
