Cell transfection

Cell transfection experiments were performed after the construction of an fmr1 overexpression plasmid using the pGEM-T/pFLAG Vector (Promega Corp., Madison, WI, USA) and transfection reagent Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The empty vector was transfected into another batch of cells in parallel. In brief, cells were inoculated in DMEM without antibiotics. When the cell density reached 90–95%, the plasmid expressing fmr1 and Lipofectamine^® 2000 were added into DMEM without FBS, and mixed gently at the ratio of DNA toLipofectamine^® 2000 of 1:2. The culture media was changed to DMEM with FBS after incubation for 6 h at 37°C with 5% CO₂. The cell transfection rates were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis after further culture for 48 h.