Fig. 4: IFI16 overexpression accelerates trilineage specification.

a–c Quantitative PCR analysis of OCT4, SOX2, SOX17, FOXA2, CXCR4, Brachyury, PAX6, and OTX2 mRNA levels in H9 cells and differentiated trilineage on Day 2 (a, endoderm; b, mesoderm; c, ectoderm; n = 4 in each group) infected with IFI16 or NC. The relative mRNA level of each gene was calculated relatively to which in H9-NC group. d–f Representative immunoblots of total lysates from H9 cells and differentiated trilineage on Day 2 (d, endoderm; e, mesoderm; f, ectoderm) infected with IFI16 or NC and probed with the antibodies for OCT4, SOX2, SOX17, FOXA2, Brachyury, PAX6, and OTX2. β-actin serves as a loading control. g Flow cytometric analysis of SOX17⁺/FOXA2⁺, Brachyury⁺/CXCR4⁺, and PAX6⁺/Nestin⁺ population in differentiated trilineage on Day 2 infected with IFI16 or NC, The signals in the fourth quadrant indicate endoderm, mesoderm, or ectoderm population. The number in each quadrant means the proportion in total cell population. h Representative immunofluorescence images staining with antibodies against OCT4, SOX2, SOX17, FOXA2, Brachyury, SNAI2, and PAX6 in the differentiated trilineage on Day 2 infected with IFI16 or NC. Upper rows, endoderm; middle rows, mesoderm; bottom rows, ectoderm. DAPI serves as a nucleus indicator. Scale bar, 200 µM. ENDO, endoderm; MESO, mesoderm; ECTO, ectoderm. NC, negative control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way ANOVA with Tukey’s post hoc test (a–c). ***P < 0.001 versus H9-NC. ^#P < 0.05, ^###P < 0.001 versus H9-ENDO, H9-MESO, or H9-ECTO.