Fig. 6

Loss of MPI stabilizes p53 through activation of the hexosamine biosynthetic pathway.

(A) Measurement of Fru6P levels showed accumulation of Fru6P in siMPI MEFs compared to NC control. Values shown are normalized to total protein. p-Value based on paired ratio two-tailed Student’s t-test. (B) Representative phenotypes of 4 dpf zebrafish larvae following injections of std MO or mpi MO and treated with 20 μM of HBP inhibitor (DON) are shown. Enlarged (left) and group (right) images are shown. (C) Distribution of the morphological phenotypes was quantified in 4 dpf mpi morphants and std MO control injected embryos treated with or without DON. Morphological rescue was observed, as an increase in embryos with normal morphology in the presence of the inhibitor, compared to mpi morphants. Two-tailed Fisher’s exact test with the Bonferroni correction for multiple comparisons, with alpha = 0.017. (D) Mpi activity assay of 4 dpf zebrafish larvae injected with std MO or mpi MO and treated with 20 μM of DON showed that Mpi activity was not significantly affected by DON treatment. Two-tailed Student's t-test was applied with Bonferroni correction for multiple comparisons, with alpha = 0.0125. (E) Representative western blot for p53 protein in 4 dpf zebrafish larvae injected with std MO or mpi MO and treated with 20 μM DON showed a decreased p53 expression in mpi morphants treated with DON, compared to non-treated. DON treatment did not induce increase in p53 in std MO. Representative western blot for three independent clutches. (F) Western blot showing levels of Gfpt1 and p53 in 4 dpf zebrafish embryos injected with either std, mpi, gfpt1, or mpi MO + gfpt1 MO. Quantified relative to tubulin loading control, and compared to std MO. Representative western blot for four independent clutches.