Experiments were performed with NP cells from non-degenerate IVDs and degenerate IVDs (NP and annulus fibrosus (AF) cells) within passage 1 to 2. Twenty thousand cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry experiments following treatment. Three hundred thousand cells were seeded in 6-well plates (Sarstedt, TC plate 6-well, Cell+, F) for ELISA and RNA extraction following treatment. All cells were left to adhere for 12 to 24 h and then serum-starved in DMEM with 1X insulin-transferrin selenium (ITS, Thermo Fisher, Waltham, MA, USA) for 6 h prior to treatment. To examine the effects of different treatments, healthy cells were treated with either 100 ng/ml Pam2CSK4 (TLR-2/6 agonist, Invivogen), 100 ng/mL Pam3CSK4 (TLR-1/2 agonist, Invivogen), or 5 μg/mL lipopolysaccharide (LPS) (TLR-4 agonist, Invivogen) for 6, 12, 24, and 48 h. Cells were either left untreated (negative control) or treated with 100 ng/mL of Pam2CSK4 for 48 h of which treatment with 100 μM o-vanillin (Sigma-Aldrich, Oakville, ON, Canada) was initiated in the last 6 h of incubation [18, 23, 30].

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