Fig. 3: IFI16 knockdown by sh1865 inhibits trilineage specification.

a–c Quantitative PCR examination of OCT4, SOX2, KLF4, SOX17, FOXA2, CXCR4, Brachyury, PAX6, and OTX2 mRNA levels in H9 cells and differentiated trilineage (a, endoderm; b, mesoderm; c, ectoderm; n = 4 in each group) infected with sh1865 or shNC. The relative mRNA level of each gene was calculated relatively to which in H9-shNC group. d–f Representative immunoblots of total lysates from H9 cells and differentiated trilineage (d, endoderm; e, mesoderm; f, ectoderm) infected with sh1865 or shNC 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 infected with sh1865 or shNC, 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 infected with sh1865 or shNC. 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 < 0.05, **P < 0.01, ***P < 0.001 versus H9-NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus H9-ENDO, H9-MESO, or H9-ECTO.