## 2 Materials and Methods
### Western Blot  
Total protein was collected from HGK and HGF with or without treatment of _E. coli_ LPS and/or hypoxia as described under Section 2.3 (Table 1). The cells were then incubated \begin{table}
\begin{tabular}{c c c} \hline \hline
**Gene** & **Primer Sequence (5/-3/)** & **Amplicon2 Size (bp)** \\ \hline TLR 1 [3, 4] & F. CAGGATAACAACAAGCCATATGTG & 238 \\  & R: GATAGTGCTTTAGGAACG & \\ \hline TLR 2 [3] & F:GTAGTGTGTGTGAAGC & 727 \\  & R: AAMCTGATCTCTGCAGTTCC & 132 \\  & F: AGTCTCTCCCAGATGTTTGCT & 405 \\  & R: GATTGCAGGTTCAGAATTCGT & 70 \\  & R: ATGATTCTCTGCAGTTGAATG & 159 \\  & TLR 5 [3] & F: GATTCCAGGAAAGATGT & 341 \\  & R: GATTCCTTTTCCTGT & 41 \\  & R: TCCCTGATGCTCTCTGTCTC & 164 \\ \hline TLR 3 & F: TTCTTGGATTGAGAGTGTGTGA & 335 \\  & R: GATTCTGAATTGGATTGGATT & 92 \\  & R: GATTCTCTGTGTGT & 92 \\ \hline TLR 3 [4] & F:CAAGAAGTTGAGTGTGTGT & 277 \\  & R: GATTCCCCACACACCTCTCT & 284 \\  & R: TCCCAGACTCACAAATCTCTCT & 686 \\  & R: GATTGGATTGGATTGGATT & 428 \\  & R: GATTATTCCAGAATCAGATC & 98 \\  & R: GATTATTGGATTGGATT & 117 \\  & R: GATTGGATTGGATT & 183 \\  & R: GATTGTGTGTGTGTGTGGGG & 151 \\  & R: GATTGGATTGGATT & 151 \\  & R: GATTGGATTGGATT & 152 \\  & R: GATTGGATTGGATT & 387 [26] \\  & R: AAGGTGGAGGAGGGTGGTG & 387 [26] \\  & R: AAGTCAAAGAGATTGG & 182 [27] \\ \hline \hline \end{tabular}  
* RT-PCR and RT-qPCR protocol described in section 2.5. 2 Amplicons were systematically sampled for sequencing to confirm that the corresponding products were, in fact, amplified from the gene fragment concerned, hence the specificity of the primer pairs used (data not shown). 3 Primers for RT-PCR. 4 Primers for RT-qPCR.  
\end{table} 
Table 2: List of primer sequences and expected product size for TLR and NLR detection 1.  
with lysing buffer (2 uL/mL protease inhibitor (P8340, Sigma-Aldrich, St. Louis, MO, USA) in 1% Triton-X100-150 mM NaCl-20 mM Tris HCl, pH 7.4) on ice for 10 min, followed by centrifugation at 12,000\(\times\)_g_ at 4 \({}^{\circ}\)C for 30 min. The supernatant was collected, then the protein concentration was measured by [PERSON](tm) BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Fifty micrograms of cellular proteins extract were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by electro-transteral to polyvinylidene dilluoride membrane (Immobilon, Sigma-Aldrich, St. Louis, MO, USA). After blocking by 5% non-fat milk at r.t. for 30 min, primary antibodies against TLR1/2/4/5/6, NOD1/2 or NLRP1/2 (mouse anti-human: TLR1, TLR4 and NLRP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TLR2 (Imgenex, San Diego, CA, USA), TLR5 (Abnova, Taipei, Taiwan), rabbit anti-human: TLR6, NLRP1 and NOD2 (Thermo Fischer Scientific, Waltham, MA, USA), NOD1 (R&D systems, Inc., Minneapolis, MO, USA); at antibody concentration: TLR1/2/4, NOD1/2 and NLRP1/2: 1:500, or TLR5/6: 1:2000) in blocking buffer (5% non-fat milk in 1% Tween 20-Tris-buffered saline, pH 7.6) was added to the blot at 4 \({}^{\circ}\)C, overnight. The membranes were then washed by 1% Tween 20-20 mM Tris-buffered saline (pH = 7.6) r.t., 3\(\times\), 5 min, and then incubated with the corresponding secondary antibody (1:2000, [PERSON] goat anti-mouse/rabbit IgG, Thermo Fisher Scientific, Waltham, MA, USA) at r.t. and visualized by an enhanced chemiluminescence system (ECL) (GE healthcare, Lifescience, Marlborough, MA, USA) at r.t. via undisturbed immersing in 10 mL ECL buffer, 22 uL p-coumamic acid, 50 uL luminol and 3 uL H\({}_{2}\)O\({}_{2}\) for 90 s [26]. \(\beta\)-actin (primary antibody, mouse anti-human, Thermo Fisher Scientific, Waltham, MA, USA, at 1:500, in 5% non-fat milk in 1% Tween 20-Tris-buffered saline, pH 7.6) was used as an internal control. HGK/HGF TLRs or NLRs blot densities from cells incubated under normoxia for 24 h were normalized with reference to \(\beta\)-actin controls, followed by semi-quantitative determination of the corresponding DAMP sensor from the same cell type after LPS or hypoxia treatment [29].