Fig 2: CREB3L1 deficiency led to substantial changes in chromatin accessibility and gene expression profile concerning protein production and secretion. a CREB3L1 acted as a transcriptional regulator by translocating to the nucleus during differentiation induction. Scale bar = 20 μm. b Heatmaps show the density of NFR summit-centered ATAC-seq signals in the Ctrl group and the sgCreb3l1_A8-4 group after 5 days of differentiation induction, respectively. The dot plot depicts the ATAC-seq outcomes, while the Gene Ontology (GO) enrichment analyses provided insight into the biological processes (c) and description of “Mouse Phenotype Single KO” (d) associated with the lost nucleosome-free regions (NFRs) in the sgCreb3l1_A8-4 group after 5 days of differentiation induction. e, f Visualization of ATAC-seq results for the Ctrl or sgCreb3l1_A8-4 cells after 5 days of differentiation induction from the UCSC Genome Browser. The blue box shows the closed NFRs in D5_sgCreb3l1_A8-4 near Dmp1 and Dspp. g Scatter plots show the significantly up-regulated or down-regulated genes identified by bulk RNA-seq analysis in D5_sgCreb3l1_A8-4. h The dot plot displays the GO enrichment for the downregulated genes in D5_sgCreb3l1_A8-4 from the bulk RNA-seq results. i The heatmap visualizes the differential expression of genes in specific GO terms identified by the bulk RNA-seq analysis

Fig 3: Deficiency of CREB3L1 resulted in a significant decrease in the level of Dmp1 and Dspp in the first molar. a The RNAscope staining of Dmp1 in odontoblasts of the first molar of WT and cKO mice at PN 3 W. Scale bar = 100 μm. b The RNAscope staining of Dspp in odontoblasts of the first molar of WT and cKO mice at PN 3 W. Scale bar = 100 μm

Fig 4: Observation of odontoblast-specific makers in incisors of DT-injected DTR-mice and Col1 (2.3)-Cre/DTR-mice after performing a series of measurements of nociceptive scores. Immunofluorescence analysis using DT-administered DTR-mice (as control; left panels) showed that DSPP-positive cells (red) were localized in both the cell-rich zone (CZ) and odontoblast (OB) layer in proximity to dentin (D). Nestin-positive odontoblasts (blue) were also observed in both CZ and OB (left panels). Analysis using Col1 (2.3)-Cre/DTR-mice (right panels) showed that DSPP-positive (red) and nestin-positive cells (blue) were not observed in the OB layer. Scale bars, 20 μm.

Fig 5: TMEM30B deficiency impaired the odontoblastic differentiation capacity of mDPCs and reduced their intracellular and supernatant protein levels. a The mRNA expression level of Tmem30b was down-regulated in the siTmem30b group compared to the Ctrl group. b After 5 days of differentiation induction, the expression of TMEM30B, DMP1, and DSPP was reduced in mDPCs treated with siTmem30b when compared to the Ctrl group. c Quantification of the relative protein expression levels of b. d Alizarin red S staining was performed to observe the calcium nodules in both Ctrl and siTmem30b groups after 14 days of differentiation induction. Scale bar = 1 cm, scale bar = 100 μm. e Design of sgRNAs for Tmem30b knockout. f Genotyping results of the homozygous knockout cell line, sgTmem30b_60. g The total amount of intracellular proteins and the percentage of decline to the Ctrl group. h The total quantity of extracellular supernatant proteins and the percentage of decline to the Ctrl group. i The protein degradation rates were measured after 24 h of treatment with 1 μg/mL cycloheximide in both the Ctrl and sgTmem30b_60 groups. n = 3. ns, not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1