Immortalized Rat NP Cell Response to FSS

An immortalized rat NP cell line used in this study was described in Oh et al. (2016) (link). The cells were cultured in Dulbecco’s Modification of Eagle’s Medium (10-013-CVR; Corning, United States) containing 10% FBS (10099-141; Gibco, Australia) supplemented with 1% penicillin-streptomycin (SV30010; Hyclone, United States) at 37°C with 5% CO₂. FSS experiments were conducted as previously described (Yang et al., 2019 (link)). Cells were seeded onto collagen I-coated culture slips (75 mm × 25 mm × 1 mm; FFCS-C; Flexcell, United States) at a density of 3.0 × 10⁴/cm² and incubated in a 5% CO₂ incubator at 37°C. When cells reached up to 85% confluence, the slips were then placed in a parallel plate flow chamber of Streamer^® System (STR-4000; Flexcell, United States) (Figure 1B) and cells are exposed to 12 or 24 dyne/cm² FSS for 0, 1, 2, 3, and 4 h. For certain experiments, NP cells were pre-treated with 10 μM CoPP (Sigma, United States, C1900) for 1 h or 500 nM rapamycin (Selleck, United States, S1039) for 12 h before exposure to FSS.