Fig. 7
Transcriptome analysis of KO and WT NPC by RNA-seq. RNA-seq was conducted using three pairs of biological replicates of WT and KO NPCs. A Heatmap of 55 genes with the lowest FDRs among 106 genes with FDR < 0.05 in NPC RNA-seq. Results of 3 biological replicate pairs (WT and KO) of NPCs are presented. Color represents the standard deviation beyond the normalized mean of each gene in all the samples tested (i.e., 3 WT and 3 KO NPC samples). B A volcano plot depicting fold changes and FDRs of mRNA levels of expressed genes in KO versus WT NPCs according to RNA-seq. The dashed vertical lines indicate log2 fold changes (twofold increase or decrease), and the dashed horizontal line marks FDR = 0.05. The names of some prominently changed genes with the lowest FDR or biggest fold changes are indicated. C Log10 fold changes of all the 111 transcripts from 106 genes with FDR < 0.05. The percentages of transcripts with upregulation and downregulation are shown. D GO analysis in terms of biological processes for the genes with FDR < 0.05. Fifteen terms with high relevance to NTD were selected out of 28 significant terms. The number of the FDR significant genes belonging to a particular term in the GO databank is indicated. More detailed information about this GO analysis, such as the p-values of the terms after Bonferroni correction and the names of genes associated with each term, are presented in SI-Table 2. E Nuclear run-on validation of four genes with significant increase and four genes without change in the RNA-seq of KO NPCs. The number of repetitions is indicated. F Reduced Folh1 RNA-seq read density in KO adrenal glands. Folh1 RNA-seq read density tracks of three pairs of WT and KO adrenal glands are illustrated. G Reduced Folh1 mRNA levels (mean ± SD) in the KO adrenal glands (left panel) and NPCs (right panel) according to RT-qPCR. The number of repetitions is shown. H Decreased FOLH1 protein levels in the intestine of KO mice according to immunoblotting. A representative blot is shown. The relative ratios (mean ± SD) of FOLH1 versus β-actin signals of KO and WT intestines based on three independent experiments are shown in the bar graph. *p < 0.05; **p < 0.01; ***p < 0.001 (paired two-way Student’s t tests)