Fig 2: Role of NFIC-miR-200b-Ambra1 pathway in modulating autophagy and apoptosis in NTDs.A schematic diagram depicting the overexpression of NFIC-enhanced miR-200b transcriptional activity, followed by the suppressed translation of Ambra1, which is the downstream target of miR-200b, leading to impaired autophagy/excessive apoptosis and the subsequent formation of NTDs.

Fig 3: miR-200b rescues NFIC knockdown-induced autophagy.A, B Protein expression of LC3 in normal and ATRA-induced NTD embryos (n = 3). C, D Protein levels of Ambra1, LC3, and NFIC after C17.2 cells were transfected with the NFIC plasmid or the control vector. E, F Effect of NFIC SiRNA on the protein expression of Ambra1, LC3, and NFIC. G, H Ambra1, LC3, and NFIC protein expression in cells transfected with the NFIC SiRNA and the control mimic or the miR-200b mimic. I The fluorescence intensity of LC3 in cells transfected with the NFIC SiRNA and the control mimic or the miR-200b mimic, scale bar = 5 µm. Experiments were repeated three times. *P < 0.05 vs. control group.

Fig 4: BioID-mediated proximity-labeling identifies DTX2-associating partners(A) BioID was fused to the N-terminus of full-length DTX2 and expressed in HeLa cells. In the presence of biotin, BioID labels nearby proteins on lysine (K) residues. Biotinylated proteins are then pulled down by streptavidin-coupled beads and analyzed by mass spectrometry (MS). Each group was done in triplicates.(B) Proteins identified in the BioID-DTX2 group were compared with those for BioID alone in a volcano plot graphed using Perseus. Red dots indicate highly enriched proteins (FOT>3, refer to STAR Methods).(C) Molecular function analysis was performed using the FUNRICH software to analyze the proteins enriched in the BioID-DTX2 group from (B).(D and E) HeLa cells treated for 24 h with different concentrations of the Notch inhibitor DBZ were harvested to assess the relative mRNA levels of HES1, DTX2, and hTERT (D) and telomerase activities (E). Data were shown as mean ± SD (N = 3). p values were calculated using two-way ANOVA. ***p< 0.001; n.s., not significant.(F and G) HeLa cells overexpressing DTX2 or NF-I family proteins were assessed for relative hTERT mRNA levels (F) and telomerase activities (G). Data were shown as mean ± SD (N = 3).(H and I) DTX2 KO cells overexpressing DTX2 or NF-I family proteins were assessed for relative hTERT mRNA levels (H) and telomerase activities (I). Data were shown as mean ± SD (N = 3). For panels (F–I), p values were calculated using Student’s t test. *p< 0.05; **p< 0.01; n.s., not significant. See also Figures S4, S5, and Table S3.

Fig 5: Expression of miR-200b and NFIC in a mouse model of ATRA-induced NTDs.A Relative expression of miR-200b in normal and ATRA-induced NTD embryos at E9.5 (n = 8). mRNA levels of NFIC (B) and NFIB (C) in E9.5 embryos from control and NTD groups (n = 8). D Protein abundance of NFIC in E9.5 mouse embryos (n = 9). E RNA expression correlation analysis for miR-200b and NFIC in NTD mouse embryos, as shown in the panel. The bar graph shows quantitative data from all experiments, which were independently performed in triplicate. *P < 0.05 vs. control group, “ns” represents no significance, compared to the control group.