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.