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. 2023 Apr 11;14:2026. doi: 10.1038/s41467-023-37703-6

Fig. 2. The effect of FOXI3 variants with in silico prediction and in vitro experiments.

Fig. 2

A The 18 identified variants on FOXI3. The predictions of the variants are classified by bins and shown on the top of each variant. The variants are classified by the type of inframe_deletion (square), frameshift (hexagon), missense (circle), and truncating (star), and the inheritance mode of autosomal dominant (blue) or with reduced penetrance (purple), autosomal recessive (green), and unknown (white). The variants of p.(Arg238Gln), p.(Arg240Cys), and p.(Ser373_Thr376del) are detected in two probands and the rest are in one proband. B Effects of the 18 variants on the transactivation of FOXI3 in HEK293T cells (n = 4/group). Gray Bars: intrinsically disordered regions (IDRs), orange bars: forkhead domain (FHD), blue bar: nuclear localization signal (NLS). *P < 0.05, **P < 0.01, ***P < 0.001. Each experiment was repeated at least three times. C The localization and distribution of wild type or mutant FOXI3-EGFP fusion proteins in HEK-293T cells. DAPI counterstain (blue) shows the location of the nucleus. EGFP (green fluorescent protein) shows the subcellular localization of the FOXI3-EGFP fusion protein. Wild-type FOXI3 is almost completely transferred into the nucleus and uniformly distributed. A majority of FOXI3 with mutant NLS is blocked outside the nucleus; FOXI3 with mutant FHD tends to form intranuclear aggregation; most of FOXI3 with mutant IDR is similar to Wild-type FOXI3. Scale bar = 10 μm. Each experiment was performed in triplicate and repeated at least three times. D Representative western blots of FOXI3-EGFP with mutant NLS. Immunoprecipitation on the EGFP-tagged FOXI3 using antibody of EGFP and detect the amount of FOXI3-EGFP in the nucleus and cytoplasmic fractions of HEK293T cells (n = 3 biologically independent experiments). Compared to the wild type, the mutant FOXI3-EGFP fusion protein is significantly trapped in the cytoplasm and the amount of mutant FOXI3-EGFP is significantly reduced in the nucleus. Significant differences between two groups (wild type and each mutant) were determined by unpaired Student’s t-test (two-tailed). Data are presented as the means ± s.d. Source data are provided as a Source Data file.