Dmem 10 013 cvr

CCK-8 (Cat # 96992) was purchased from Sigma (St. Louis, MS, USA). Fetal bovine serum (FBS) was purchased from Ausbian (Sydney, Australia). DMEM (10-013–CVR) was purchased from Corning Inc. (Corning, NY, USA). Trypsin (Cat # T4665) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). D-Hanks Hanks was obtained from Shanghai GeneChem Co. Ltd. (Shanghai, China). 4-hydroytamoxifen (4-OH-TAM) was purchased from Sigma (St. Louis, MS, USA). TRIZOL RNA Isolation Kit (Catalog #: 12183555) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). M-MLV (M1705), dNTPs (U1240) and RNase inhibitor (N2115) were obtained from Promega (Madison, WI, USA). Oligo dT (B0205) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Bulge-LoopTM miRNA qPCR Primer Sets were synthesized by Ibibio (Guangzhou, Guangdong, China). Reverse and forward primers were synthesized by Shanghai Genechem Co., LTD (Shanghai, China). SYBR Master Mixture (DRR041B) was obtained from TAKARA (Daliang, Liaoning, China). Reagents for reverse transcription were purchased from Axygen (Union City, CA, USA).

Neonatal SD rats were purchased from Nantong University, and their sciatic nerves were extracted for cell collection. Cells were treated with anti-Thy1.1 (1:1000, M7898, Sigma, St. Louis, MO, USA) and rabbit complement (Invitrogen, Carlsbad, CA, USA) to remove fibroblasts. Purified Schwann cells were cultured in DMEM (10–013-CVR, Corning, NY, USA) containing 10% FBS (10099141c, Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (c0222, Beyotime, Shanghai, China), 2 μM forskolin (Sigma), and 10 ng/ml HRG (R&D Systems Inc., Minneapolis, MN, USA). Cultured Schwann cells were transfected with siRNAs against MOGS (siRNA-1: RibiBio, siG2010140505022069, GUCUAUUUCGGCAUGAAGA, siRNA-2: RibiBio, siG2010140505023161, UCGGCAACAUAUCUAUGAU, and siRNA-3: RibiBio, siG2010140505024253, GUAAAGAGCCACCUAAACA) or a random sequence non-targeting negative control (RibiBio, Guangzhou, Guangdong, China) using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

The BEL-7404 and SMMC-7721 cells were infected with the lentiviral vector containing the shRNA against RSPH14 (KD group) or the negative control vectors (NC group) for 3 days. Then, the infected cells were seeded into 96-well plates (2000 cells/well) and incubated at 37 °C with 5% CO₂ in DMEM (10-013-CVR, Corning Inc., Corning, NY, USA) with 10% FBS (VS500T, Ausbian). A BrdU kit (11647229001, Roche Applied Science, Penzberg, Germany) was used to evaluate cell proliferation, according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate reader (M2009PR, Tecan Group Ltd., Mannedorf, Switzerland).