Plasmids
Wildtype EGFP-CIC plasmid and the R353X EGFP-CIC mutant plasmid were reported in our previous paper (Cao et al., 2021). Variants identified in NTDs were introduced into wildtype EGFP-CIC plasmid using site direct mutagenesis method by Genescript company.
Cell culture and transfection Hela cells and NIH3T3 cells were grown in Dulbeco’s Modified Eagles’s Medium (DMEM, Sigma, D6429), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1X Antibiotic-Antimycotic (Gibco). NIH3T3 cells were cultured on 0.1% Gelatin (Gibco)-coated plate. Cell cultures were maintained at 37℃ in a humidified atmosphere containing 5% CO2. Transfection was performed at 50-70% confluency using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Medium was replaced with the culture medium without antibiotics prior to transfection. Further experiments were performed 24-48 hours after transfection.