Fig 2: Subcellular localization and expression of TGFBI in transfected HEK293T cells. (a) Immunofluorescence staining showing localization of wild-type (WT) and mutant (p.S77X) TGFBI. Cells were harvested 48 h post-transfection. TGFBI was detected using an anti-His tag antibody (green); nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. (b) Western blot analysis of TGFBI expression levels in empty vector, wild-type TGFBI, and p.S77X groups. β-actin served as a loading control.

Fig 3: Flow cytometric analysis of apoptosis and Western blot analysis of caspase-3 in transfected HEK293T cells. (a,b) Representative flow cytometry plots and quantitative analysis of Annexin V-FITC/PI staining in empty vector, wild-type TGFBI (WT), and p.S77X TGFBI(S77X) groups at 48 h post-transfection. Quadrants indicate viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V−/PI+) populations. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001). (c) Western blot analysis of caspase-3 and cleaved caspase-3 expression in the three groups at 48 h post-transfection. β-actin served as a loading control.

Fig 4: Identification of the heterozygous variant of TGFBI in a family with NSOFCs. (a) Pedigree of the family with NSOFCs. The arrow indicates the proband (D1). Filled symbols represent affected individuals (D1 and D2) with NSOFCs. (b) Clinical photographs of the lip, palate, and alveolus from subjects D1 and D2; (c) Sanger sequencing chromatograms confirming the heterozygous c.230C > A (p.S77X) variant in TGFBI. Arrows indicate the mutated loci.

Fig 5: Transcriptomic and Western blot analysis of p38-MAPK signaling. (a) Volcano plots of differentially expressed genes (DEGs) among p.S77X, WT TGFBI, and vector groups (p.S77X vs. WT, WT vs. vector, and p.S77X vs. vector). Significantly upregulated and downregulated genes (adjusted p ≤ 0.05) are shown in red and blue, respectively; non-significant genes are in gray. (b) Gene Ontology (GO) enrichment analysis of apoptosis-related terms among DEGs. Enrichment significance is represented as −log10 (adjusted p-value). (c) KEGG pathway enrichment analysis of apoptosis-related DEGs. Rich factor indicates the degree of enrichment, dot size reflects gene count, and dot color denotes adjusted p-value range. (d) Heatmap showing MAPK-related gene expression profiles across vector, WT TGFBI, and p.S77X samples. Each column represents a sample; each row represents a gene. Expression levels are color-coded from blue (low) to red (high). (e) Western blot analysis of total p38 and phosphorylated p38 (p-p38) in empty vector, WT, and p.S77X groups at 48 h post-transfection. β-actin served as a loading control.