TRRAP silencing destabilizes mutp53 via the MDM2-proteasome axis. (A) Experimental design. Namalwa cells were transduced with an IPTG-inducible shRNA against TRRAP (shRNA #233) or a nontargeting control (NT). 48 to 96 hours after induction, cells were lysed and either collected immediately (total protein) or subjected to immunoprecipitation (IP) with antibodies against p53 or IgG (negative control) before mass spectrometry. The experiment was performed in biological triplicates. (B) Volcano plot visualizing differential total protein expression 96 hours after TRRAP knockdown compared with a nontargeting control (NT). Proteins denoted in black were significantly differentially expressed (adjusted P < .05, |log2FC| > 0.5). p53 is highlighted. P values were calculated using empirical Bayes statistics on protein-wise linear models. (C) Gene set enrichment analysis of differentially expressed proteins from panel B. Upregulated gene sets are denoted in red, downregulated in blue. Analysis was performed using PAGE with the MSigDB gene set collection “Hallmark.” Only significantly altered gene sets are shown (adjusted P < .05). (D) Heatmap of mutp53-bound proteins (proteins significantly enriched in p53 IPs compared with IgG control IPs). Values denote hierarchically clustered log2 fold changes of protein abundances in TRRAP-silenced samples compared with control knockdown (NT) samples. The cellular localization⁴⁴  and known p53 interactions⁴²  are indicated. Numbers within the clusters indicate the number of proteins. TP53, EIF3F, and XPO1 are highlighted. (E) Rescue of the TRRAP-silencing–mediated mutp53 degradation by treatment with nuclear export inhibitors. Namalwa cells were transduced with an IPTG-inducible shRNA against TRRAP (shRNA #233) or a nontargeting control (NT). 48 hours after induction, cells were treated with DMSO (0.005%), leptomycin B (20 nM), selinexor (500 nM), or verdinexor (500 nM) for 14 hours before they were subjected to p53 flow cytometry. Values denote p53 MFI normalized to DMSO-treated cells transduced with the NT. (F) Rescue of the TRRAP-silencing–mediated mutp53 degradation by treatment with the MDM2 inhibitor Nutlin-3a. Namalwa cells were transduced with a shRNA against TRRAP or a non-targeting control (NT). 4 days after transduction, cells were treated with DMSO (0.1%) or Nutlin-3a (10 µM) for 48 hours before they were subjected to p53 flow cytometry. Values denote p53 MFI normalized to DMSO-treated cells transduced with the NT. (G) Rescue of the TRRAP-silencing–mediated mutp53 degradation by CRISPR-Cas9–mediated knockout (KO) of MDM2. KO cells were transduced with a shRNA against TRRAP or a nontargeting control (NT) and subjected to p53 flow cytometry 4 days after transduction. Values denote p53 MFI normalized to control (GFP) KO cells transduced with the NT. KOs were generated by transduction of Namalwa cells with constructs harboring both Cas9 and sgRNAs against MDM2 or GFP (negative control).