## 2 Materials and Methods
### MTT Assay  
To compare the growth rates of HGK and HGF cultured in 1% or 18% O\({}_{2}\), HGK or HGF were seeded at a density of 3.0 \(\times\) 10\({}^{3}\) per well in 96-well plates (Table 1). Wells with evenly distributed cells were used. After 24 h culture, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) assay was performed according to the manufacturer's protocol (ATCC, Manassas, VA, USA). Briefly, HGK or HGF (treated or untreated) were incubated with 10 \(\mu\)L MTT for 2 h and then the MTT reagent was replaced with 100 \(\mu\)L dimethylsulfoxide for another 2 h. Absorbance values for each well were measured by a spectrophotometer at 570 nm (Bio-Rad Model 3550, Bio-Rad, Hercules, CA, USA). For LPS treatment, from a stock solution of 1 mg/mL _E. coli_ LPS in Dulbecco's phosphate-buffered saline, the correct amount of the endotoxin was added to the cell wells to achieve a final concentration of 2 \(\mu\)g/mL at 18% O\({}_{2}\), 24 h [28].  
All experiments were repeated three times at separate occasions, using HGK/HGF explants at the third passage.  
Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Reverse Transcription PCR (RT-Qcr) Analysis  
Total RNA was purified from treated or untreated HGK or HGF and HGT biopsies (Table 1) via disruption and homogenization, using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The human acute monocytic leukemia cell line (THP-1) was used as the positive control for TLR. HGT were maintained in RNAlater at 4 \({}^{\circ}\)C overnight before proceeding with RNA extraction.  
For RT-PCR, cDNA was synthesized from 1 \(\mu\)g of total RNA from each sample, using Superscript First-Chain Synthesis Kit (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed using KAPA2G Fast HotStart Readymix (Kapabiosystems, Boston, MA, USA), and two-step RT-qPCR was carried out using cDNA from control/treated cell explants by TaqMan Real-Time PCR Master Mix (Invitrogen, Carlsbad, CA, USA) according to the protocol suggested by the manufacturer. The RT-PCR amplification process was performed at initial denaturation for 5 min at 95 \({}^{\circ}\)C and then 40 cycles of 95 \({}^{\circ}\)C for 15 s, 56-61 \({}^{\circ}\)C for 15 s, and 72 \({}^{\circ}\)C for 5 s. For RT-qPCR amplification, the initial denaturation was for 10 min at 95 \({}^{\circ}\)C, 40 cycles of 95 \({}^{\circ}\)C for 15 s, and 60 \({}^{\circ}\)C for 1 min. GAPDH (RT-PCR) and \(\beta\)-actin (RT-qPCR) were utilized as internal controls [26; 27]. The primer sequences used are shown in Table 2. Target mRNAs/transcript levels of HGK/HGF under normoxia (18% O\({}_{2}\)) at 24 h; hypoxia (1% O\({}_{2}\)) at 24 h; and ug/mL _E. coli_ LPS challenge under normoxia at 24 h were determined with reference to the internal control (\(\beta\)-actin). Then, the corresponding relative target transcript levels under various test conditions were normalized against that of the same cell type under normoxia at 24 h.  
Except HGT biopsies, all cells were repeatedly cultured, treated and RNA harvested three times for the RT-PCR and RT-qPCR experiments. Randomly selected positive amplicon samples, representative of all ten DAMP sensors of interest, were sequenced to confirm the identity of the related cDNA concerned. This was repeated systematically in all three independent repetitive experiments.