Fig 2: (a) Hematoxylin and eosin stain (40x magnification) from the first, atypical meningioma showed a highly cellular tumor with large nuclei and prominent nucleoli, and an increased mitotic count. (b) The tumor showed a varying expression pattern for EMA (20x magnification). (c) SSTR2A showed diffuse and strong membranous and cytoplasmic positivity (20x magnification). (d) MDM2 amplification resulted in MDM2 overexpression, since additional immunohistochemistry showed nuclear positivity for MDM2 (40x magnification).

Fig 3: RITA restores TP53 activity in medulloblastoma cell lines with and without TP53 mutations(A) TP53 and CDKN1A protein expression after 48 h of RITA treatment assessed by western blot. Ethanol served as a negative control. wt = wildtype, mut = mutation. (B, C) Relative expression of CDKN1A and MDM2 mRNA after 48 h of RITA treatment measured by real-time polymerase chain reaction (PCR). Ethanol served as a negative control. * = P < 0.05 in Student's t-test.

Fig 4: FoxO3a is downregulated in TamR BBCs. (A) FoxO3a transcripts were analyzed by real-time PCR in growing MCF-7 and TamR cells. Each sample was normalized vs. its 18S rRNA content and presented as fold enrichment versus MCF-7. The results represent the mean ± s.d. of three independent experiments. *, p < 0.01 vs. untreated. (B) Cytoplasmic and nuclear protein extracts from a duplicate set of cells were subjected to WB (30 µg/lane) to evaluate the subcellular localization of FoxO3a. GAPDH and Lamin B (cytosolic and nuclear markers, respectively) were used as loading controls and to assess the quality of the subcellular protein fractionation. (C) Immunostaining of FoxO3a expression and localization (green) in MCF-7 and TamR growing cells; nuclear integrity was visualized by DAPI (blue) (400x magnification) (D,E) Comparison between AKT (D) and MAPK (E) signal transduction pathways in MCF-7 and TamR cells. Cells were starved in PRF-SFM for 16 h and then treated or not with EGF (100 nM). Proteins were analyzed by WB, using the indicated antibodies. (F) PLA. MCF-7 and TamR cells were seeded in MW8 (Lab-Tek™ Chamber Slide System, Nunc™), left to adhere for 48 h, then starved in PRF-SFM and pre-treated with MG132 (20 µM) or left untreated (-). The next day, EGF (100 nM) was added for 30 min where indicated. Antibodies against FoxO3a and MDM2 were used to detect the active complexes. Captures of the stained FoxO3a/MDM2 complexes were analyzed by ImageJ software and their relative abundance vs. untreated samples was reported in the graph. The whole experiment, with the relative images, has been included in Supplementary Information (Figure S3). (G) A duplicate set of cells was treated as in (F) and protein expression was assessed by WB using indicated antibodies.

Fig 5: USP2 inhibits the cellular actions of p53 through Mdm2a. USP2 knockdown decreased Mdm2 expression. MyLa2000 cells were transfected with either siRNA control or USP2 siRNA for 24h, followed by treatment with 5μM nutlin3a for 24h. Western blot was used for detecting USP2, Mdm2 and p53 protein level. β-actin was used as internal control, and relative protein expression levels are reported below the corresponding western blot bands. The experiment was repeated 3 times. Representative data was shown. b. USP2 knockdown increase p53 transcription activity. The expression of p21 was used to indicate p53 transcription activity. qPCR was applied to examine p21 expression in MyLa2000 cells transfected with siUSP2/siRNA with or without nutlin3a treatment. Paired T-test was used to calculate P-value. c. A proposed model for the regulation of USP2. PUVA and nutlin3a activate p53 and promote cell apoptosis. USP2 is induced by PUVA and nutlin3a and increases cell resistance to apoptosis via modulation of Mdm2/p53 interaction, constituting an anti-apoptosis protective loop.