Figure 2
NUDT16 exerts growth-inhibitory effects and destabilizes mRNA targets that regulate MYC protein stability. (a) Top, restoration of NUDT16 protein expression upon stable transduction in MOLT-4 cells (NUDT16-OE) was associated with reduced viability in the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in comparison to empty vector-transduced cells. Probabilities are those from permutation test; middle, tumor volume monitoring of xenograft samples derived from NUDT16 MOLT-4 cells transduced with NUDT16 or the empty vector. ***P<0.001, **P<0.01. Restoration of NUDT16 expression was associated with smaller tumors; below, weight and volume of the tumors excised at 60 days. Both measures were lower in the xenografts derived from MOLT-4 derived cells transduced with NUDT16. Probabilities are those associated with Student’s t-tests. Results are presented as the mean±s.e.m., n=10. (b) Nuclear/cytoplasmic fractionation of NUDT16-methylated (CCRF-CEM, Jurkat, MOLT-4 and MOLT-16) and NUDT16-unmethylated (HL-60, K562 and KOPN-8) cell lines show expression and cytosolic accumulation of NUDT16 in the unmethylated cells. Purity of fractions was assayed with the nuclear protein β-Tubulin and the cytosolic Lamin-β. WCL, Whole Cell Lysate. (c) Left, validation of the mRNA expression levels by RT-qPCR following the Actinomycin D treatment of the RNA-immunoprecipitation-derived candidates FBXO28 and USP37 in NUDT16 or empty vector-transduced MOLT-4 cells. Restoration of NUDT16 expression destabilizes both mRNAs; middle, western blot assays confirm reduction of FBXO28 and USP37 protein expression upon NUDT16 transduction in MOLT-4 cells; right, western blot shows that the diminished FBXO28 and USP37 levels upon NUDT16 restoration are also associated with a decrease in their target, the C-MYC protein.
