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. 2020 Nov 10;11:542004. doi: 10.3389/fgene.2020.542004

FIGURE 4.

FIGURE 4

The novel GLI3 p.Gln247∗ variant blocks the SHH pathway. (A) Western blotting analysis of the ectopic expression of GLI3 wild-type and mutant plasmids in HEK293T cells. (B) Expression levels of core proteins in the SHH signaling pathway in wild-type and mutant GLI3 overexpression HEK293T cells. Expression of PTCH1 and GLI1 is lower in the HEK293T cells with the mutant GLI3 plasmid or empty vector than with GLI3FL. (C) Localization of GLI3-Flag and GLI3 p.Gln247∗-Flag in HEK293 cells by immunoprecipitation. (D) Localization of the wild-type GLI3 and p.Gln247∗ GLI3 protein in HEK293T cells. Nucleocytoplasmic separation detection of GLI3 wild-type and p.Gln247∗ protein in HEK293T cells exhibited cytoplasmic accumulation. Confocal microscopy showed that wild-type GLI3 protein localized within the cytoplasm and nucleus, and the GLI3 p.Gln247∗ variant protein was strongly detectable in cytoplasm but barely in the nucleus. To quantitatively compare the intracellular localization ratio of the GLI3 wild-type and p.Gln247∗ protein in the nucleus and cytoplasm, fluorescence intensity data was collected from three points at random in these two compartments of the each cell separately. The average nucleus/cytoplasm intensity ratio of flag fluorescence was calculated from 10 HEK293T cells expressing GLI3 wild-type and p.Gln247∗ variant protein separately. Statistical differences were analyzed by t-test. Histograms showed that the ratio of flag fluorescence distribution in the nucleus and the cytoplasm was significantly different between wild type and p.Gln247∗ transfected cells (p < 0.0001). (E) Immunoprecipitation and western blotting assays of the binding of SUFU with wild-type and mutant GLI3 protein. IB: immunoblotting.