Fig 2: Continuous MDM2-dependent proliferation of MYC- and MYCN-transformed cone precursors. (A) MYC and MYCN-transduced retinal explants at 51 and 56 DIC and co-stained for Ki67 (top) and GFP+ (bottom). Nuclei are stained with DAPI. (B) Growth curves of MYCN- and MYC-transformed cone precursors after removal from cultured retina, expansion for 30 DIC, and plating at 1×104 cells per well, and measured in three independent wells for each time point. (C) Percent live cells detected 78 h after transduction of MYCN-transformed cone precursors with shMDM2 and control shSCR shRNAs in absence or presence of 10 or 20 µM pifithrin-α (PFT-α) for the final 40 h of culture.

Fig 3: MDM2-independent expression of MYC in SHH and Group 3 medulloblastomas. Western analysis of MDM2, MYC, and a-tubulin expression in MYC overexpressed, p53 WT, SHH medulloblastoma cell lines DAOY and UW-228-2 (A), and Group 3 medulloblastoma cell lines D283Med, D341Med, and D425Med cell lines (B) at 4 days post-transduction. Immunoblots were probed for a-tubulin as loading control.

Fig 4: Inhibition of DHODH stabilizes the p53 tumor suppressor protein.a Parental 4T1 cells and D0–D25 sublines were subjected to microchip analysis and a heat map for the 119 transcripts involved in ribosome biogenesis was constructed. b–e, g MCF7 and HCT116 cells were exposed to leflunomide (LFM, 50 µM) for 72 h in the presence or absence of uridine (U; 50 µg/l). Expression of 45S rRNA (b) and 18S rRNA (c) was assessed by qRT-PCR. d Immunoblotting detection of RPS6 was performed. ß-Actin was used as a loading control. e Immunofluorescent detection of B23 in MCF7 cells was performed. f Immunoblotting detection of p53 pS15, p53, and p21 in MCF7 cells treated 72 h with LFM (50 µM) after downregulation of RPL5 and RPL11 alone or in combination using specific siRNA. Non-targeting siRNA (siNC) was used as a control. ß-Actin was used as a loading control. g Immunoblot detection of MDM2 in MCF7 and HCT116 cells. ß-Actin was used as a loading control. h Co-immunoprecipitation of p53 followed by immunoblot analysis of p53, MDM2, ubiquitinated p53, and ubiquitinated MDM2 in MCF7 control and LFM (50 µM, 72 h)-treated cells. GAPDH was used as an input control. Immunoprecipitated samples and input represent one membrane with different intensity of signal. i Levels of ubiquitinated p53 and MDM2 related to their immunoprecipitated total forms were evaluated from three independent experiments. j Co-immunoprecipitation of MDM2 followed by immunoblot analysis of MDM2, p53, and RPL5 protein in MCF7 control and LFM (50 µM, 72 h)-treated cells. GAPDH was used as an input control. Immunoprecipitated samples and input represent one membrane with different intensity of signal. k Co-immunoprecipitation of p53 followed by immunoblot analysis of total and ubiquitinated p53 and MDM2 in MCF7 cells treated with LFM (50 µM, 72 h) after downregulation of RPL5 and RPL11 alone or in combination using specific siRNA. Non-targeting siRNA (siNC) was used as a control. p53, MDM2, and ß-tubulin were used as an input control. l The level of ubiquitinated p53 related to its immunoprecipitated total form was evaluated from three independent experiments. In b, c, i, and l, data are shown as mean ± SEM, n = 3. *P < 0.05, two-way ANOVA. In other panels, representative experiment (from total number of three experiments) is shown.

Fig 5: Downregulation of MYCN but not MYC in MDM2-depleted retinoblastoma and neuroblastoma cells. (A, B) Western analysis of MDM2, MYCN, and/or MYC in MYC and MYCN-expressing and p53 WT RB176 retinoblastoma cells (A) and SK-N-BE(1) neuroblastoma cells (B) after lentiviral transduction of shMDM2-1, shMDM2-2, or the shSCR control. (C, D) Western analysis of MDM2, MYC, p53, and p21 in MYC overexpressing, TP53 wild type CHLA 255 and SH-SY5Y neuroblastoma cells transduced as in (A) with additional transduction of shSCR or shTP53 and analyzed 40 h post transfection. Relative expression normalized to a-tubulin is shown below each blot. (E) Western analysis of MDM2, MYC, and MYCN in SH-SY5Y cells stably transduced with BN-MCYN or the empty BN lentiviral expression vector. Immunoblots were probed for a-tubulin as loading control.