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Fig. 3. Nucleostemin increases the protein level of MDM2 by preventing its degradation and ubiquitylation. (A) Coexpression of nucleostemin (HA-tagged) increased the protein levels of exogenously expressed MDM2 in a dose-dependent manner. (B) Knocking down the expression of nucleostemin with nucleostemin-targeting shRNAmir constructs (shNS-1 and shNS-2) decreased the amount of MDM2 protein. (C) MDM2 protein stability, with or without nucleostemin overexpression (NS vs Ctrl), was measured in H1299 cells. After cycloheximide treatment, cell lysates were collected from 0 to 4 hours at 0.5-1 hour intervals. The amount of MDM2 protein at every time point was measured in three independent experiments, adjusted based on their -tubulin amounts, and expressed as a percentage of the MDM2 at the 0 time-point. (D) Nucleostemin depletion by shNS-2 cotransfection increased the degradation of MDM2. Protein degradation assays were performed over a 50 minute window. (E) HEK293 cells were transfected with (His)₆-tagged ubiquitin, MDM2 and/or nucleostemin (wild-type or mutant) plasmids as specified. Ubiquitylated MDM2 products were pulled down by Ni²⁺ Sepharose (His PD) and detected by anti-MDM2 (SMP14) antibody. Overexpression of wild-type nucleostemin slightly decreased the polyubiquitylation of MDM2 compared with the control sample. Overexpression of the nucleoplasmic mutants of nucleostemin (dB, G256V and dB(256)) significantly decreased the ubiquitylated products of MDM2. Sup, supernatant. (F) Conversely, depleting the endogenous nucleostemin by the nucleostemin-targeting siRNA (siNS) increased the ubiquitylation of MDM2 compared with cells treated with a scrambled siRNA (siScr).

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