All experiments were completely randomized and repeated in triplicate. Specifically, embryos from three litters each group were analyzed as we previously described (8 (link)–10 (link)). For immunostaining, two to three embryos from each litter and four to five sections per embryo were stained, and an average for signal intensity was obtained for that litter. For immunoblotting, two to three embryos from a litter in each group were combined for a single run, and there were three runs per experiment. For RT-qPCR, two to three embryos per litter were combined, and three litters per group were analyzed. For cell culture studies, experiments were repeated in triplicate as we previously described (60 (link)). Data are presented as the means ± SEs. Student’s t test was used for two group comparisons. One-way analysis of variance (ANOVA) was performed for comparisons of more than two groups using SigmaStat 3.5 software. In ANOVA, a Tukey test was used to estimate the significance. Significant differences between groups in NTD incidence expressed by the number of embryos were analyzed by the chi-square test or Fisher’s exact test using SigmaStat 3.5 software. Differences were considered statistically significant when P < 0.05.