10 013 cvr

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.

The cell lines FaDu (human pharyngeal squamous carcinoma cell) and Tca-8113 (human tongue squamous carcinoma cell) were purchased from Cell Bank of the Chinese Academy of Sciences in Shanghai and cultured in DMEM medium (10-013-CVR, Corning) containing 10% FBS (VS500T, Ausbian), 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

CMU4232 (EV71/CMU4232-1/BJ/CHN/2008) was originally isolated from a HFMD patient by our group during the 2008 outbreak in Beijing^{8 (link)}, and it was passaged in RD cells (National Infrastructure of Cell Line Resource, Beijing, China) and stocked in our laboratory. An infectious cDNA clone of EV-A71 (pSVA14-Isehara ver4) was kindly provided by Professor Satoshi Koike, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. The RD cell line was cultured in DMEM containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning, 10-013-CVR, USA) supplemented with 10% fetal bovine serum (FBS) (Corning, 35-076-CV, USA), 100 IU of penicillin, and 100 μg of streptomycin per ml. The cells were incubated at 37 ℃ and with 5% CO₂. CMU4232 and the rescued virus, CDV-Isehara were harvested from RD cultures by freezing and thawing three times and were stored at −80 ℃. The titers of the virus stocks were tested using a modified plaque-forming assay and determining the CCID50.

Primary Schwann cells were collected from sciatic nerves of neonatal SD rats, purified with anti-Thy1.1 (1:1000, M7898, Sigma) and rabbit complement (Invitrogen, Carlsbad, CA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; 10-013-CVR, Corning, NY, USA) containing 10% fetal bovine serum (FBS; 10099141c, Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (c0222, Beyotime, Shanghai, China), 2 μM forskolin (Sigma), and 10 ng/ml β-heregulin (HRG; R&D Systems Inc., Minneapolis, MN, USA) in a humidified 5% CO₂ incubator at 37 °C. For LIF or EREF knockdown, cultured primary Schwann cells were transfected with siRNA segments targeting LIF (siRNA-1 sequence: GCTCATTCTGCACTGGAAA and siRNA-2 sequence: ATGCCAATGGGACAGAGAA), a siRNA segment targeting ERFE (sequence: TGAAGGAGTTCCAGTTGTT), or a non-targeting negative control (random sequence, RiboBio, Guangzhou, Guangdong, China) for 48 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen). For LIF overexpression, cultured primary Schwann cells were transfected with LIF-overexpressing-lentivirus (pLenti-CMV-EGFP-P2A-LIF-3FLAG) or a negative control lentivirus (pLenti-CMV-EGFP-P2A-MCS-3FLAG) (OBiO, Shanghai, China) for 72 h.