## 2 Materials and Methods
### Cell Culture  
Healthy gingiva biopsies were immersed in a 1.5 U/mL dispase solution (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) in neutral Ca\({}^{2+}\)/Mg\({}^{2+}\) free phosphate buffered saline at 4 \({}^{\circ}\)C for 8 h. The epithelium was then mechanically sheared from the connective tissue. Minced epithelial and subepithelial tissues were utilized for HGK or HGF primary culture, respectively. To obtain primary HGK or HGF explants, Gingival Keratinocyte Medium (1:1 mixture of Defined keratinocyte-SFM and Epilife(r) with calcium, Life Technologies Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (FBS), or Dulbecco's Modified Eagle Medium with 10% FBS, respectively, was used. Any  
\begin{table}
\begin{tabular}{l c c c} \hline \hline
**Experiment Group** & \(n\) & **Age Range (Years)** & **Gender (f/M)** \\ \hline _Immuno-histochemistry_ & & & \\ Healthy & 13 & 18–27 & 5/8 \\ Periodontitis & 17 & 38–46 & 8/9 \\ RT-PCR1  & 16 & 14–25 & 9/7 \\ HGK/HGF explant & 27 & 16–29 & 19/7 \\ _MTL essay1_  & 9 & 19–24 & 4/5 \\ _Selected DAMP sensor transcripts (RT-qPCR) or protein detection_  & & \\ Control (18% Oz, nil E. coli LPS) & 13 & 16–25 & 8/5 \\ Hypoxia only (1% Oz, nil E. coli LPS) & 13 & 16–25 & 8/5 \\
2 ug/mL E. coli LPS only (18% Oz) & 13 & 16–25 & 8/5 \\ Hypoxia (1% Oz) and 2 ug/mL E. coli LPS challenge2 & 13 & 16–25 & 8/5 \\ \hline \hline \end{tabular}  
HGF contamination in HGK culture was removed by brief trypsinization (0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) without EDTA, 3 min), followed by reseeding the 'cleaned HGK'. The same HGK purification procedure was repeated until no spindle-shape cells were detectable under microscopy. Samples of purified, early confluent explants from each donor on 35 mm dishes were carefully confirmed by carboxyfluorescein hydroxysuccinidyl ester staining when HGK appeared as homogenous cobblestone-shaped cells, free of any contaminating spindle-shaped fibroblastic cells or HGF as homogenous spindle-shaped cells without cells of any other shapes, smaller sizes or morphology, before use. In the case of doubt, the cells concerned were not used for any experiments. HGK and HGF from individual donors were always cultured separately. The cells at third passage were harvested for in vitro experiments [26; 27].  
HGK and HGF cultured on individual 60 mm \(\times\) 15 mm plates were treated with 2 \(\mu\)g/mL _E. coli_ LPS (From O111-B4, catalog no. L3024, Sigma-Aldrich, St. Louis, MO, USA), 18% O\({}_{2}\) (normoxia), 5% CO\({}_{2}\) for 24 h. Both cells cultured on same conditions were also treated with 1% O\({}_{2}\), 5% CO\({}_{2}\) for 24 h (hypoxia). Low oxygen concentration was maintained by a hypoxia incubator (Catalog no. 3131, Forma Water-Jacketed CO\({}_{2}\)/O\({}_{2}\) incubator, Thermo Fisher Scientific, Waltham, MA, USA). The corresponding HGK/HGF from the same donor cultured in designated media without the challenges (_E. coli_ LPS or hypoxia) under standard conditions (18% O\({}_{2}\), 5% CO\({}_{2}\)) were used as controls (Table 1) [27].  
All experiments were repeated thrice on separate occasions with cell explants from different donors.