Fig 2: TP53 or MDM4 polymorphisms might alter the impact of the MDM4T454M mutation among NCI-226 family members.(A) Genotyping of polymorphisms that may affect the p53 pathway. The SNPs rs1800371 and rs1042522 modify the p53 protein sequence (28,29), whereas rs17878362 and rs17880560 are singlets (A1) or doublets (A2) of G-rich sequences in noncoding regions of TP53 that affect p53 expression (30). SNPs rs117039649 and rs2279744, in the MDM2 promoter, affect MDM2 mRNA levels (3,31). Three SNPs are at the MDM4 locus: rs4245739 in the 3'UTR region affects MDM4 protein levels (4), whereas rs11801299 and rs1380576 were associated with an increased risk of developing retinoblastoma (32), a cancer type with frequent MDM4 alterations (51). Polymorphisms that differ among family members are in bold, with the allele (or haplotype) associated with increased p53 activity in green (because it may synergize with the effects of the MDM4T454M mutation). Alleles (or haplotypes) for which there is evidence of decreased p53 activity, or for which the effect is uncertain, are highlighted in red or blue, respectively. Please note that the clinical effects of the TP53 rs1042522 SNP have recently been contested (33), so that all alleles for this SNP were labeled in blue. MAF, minor allele frequency reported for all gnomAD populations combined. https://gnomad.broadinstitute.org (52). (B) Comparative analysis of primary fibroblasts from family members 226-4 and 226-8. p21 and RTEL1 mRNAs, extracted from cells from relatives NCI 226-4 and NCI 226-8 or two unrelated patients with DC carrying a TINF2 or a TERT mutation, were quantified using real-time PCR, normalized to control mRNAs, and then expressed relative to the mean values in TINF2 and TERT mutant cells. ns, not significant, **P < 0.01 and ***P < 0.001 in a Student’s t test.

Fig 3: Forced expression of Survivin affetcs mitosis and augments polyploidy in cells with mutant p53, loss of p53 and astrocytoma cells with expression of SV40 TAg. a Representative images of U251-MG cells stained with DAPI (chromosomes) (top panel: control cells; bottom panel: Survivin cells). Abnormal interphase cells (white arrows) and increased chromosome numbers as well as lagging chromosomes are depicted (white circle and white arrows). b Relative mean percentages of U251-MG and SVGp12 cells displaying abnormal nuclei and aberrant mitoses (mean values ± SD; *p < 0.051**. p < 0.01, ***p < 0.001; Student’s t-test. c Representative FACS-assisted DNA-content analysis of U251-MG (top panel) and SVGp12 cells (bottom panel) with overexpression of Survivin compared to the empty vector transduced controls. Note the increased 4n peak and >4n populations. d Mean increase in the fraction of Survivin-transduced cells with DNA content of 4n and >4n compared to empty vector-transduced controls (mean values ± SEM). Cell lines with loss of p53 function showed increased cell fractions with DNA content =4n in contrast to cells containing wildtype p53. *p < 0.05, **p < 0.005, ***p < 0.001. All data were collected 72 h after transduction of cells

Fig 4: Western blot analysis of the four proteins in the colorectal tumors and matching normal tissue margins (control). (A) Western blot analysis of the four proteins. (B) Expression levels of the four proteins normalized to ß-actin. *P<0.001. P504S, a-methylacyl-CoA racemase; Bcl-2, B-cell lymphoma 2; p53, tumor protein p53.

Fig 5: Overexpression of Survivin in glioma cells affects cell cycle and proliferation in a p53 dependent manner. a Western blot analysis showing cell lysates from U87-MG and U87-MGshp53 after transduction with Survivin and control vectors. The membranes were blotted with anti-p53 (53 kDa), anti-p53s15 (53 kDa), anti-p21waf/cip (21 kDa), anti-?H2AX (16 kDa), anti-cyclin D1 (37 kDa) and E (42 kDa singlets +50 kDa doublets) antibodies and the signals comparing control and Survivin cells were measured. The relative band density (fold increase) obtained from the densitometric analysis and normalized to the corresponding control is depicted. a-actin (42 kDa) was used as an internal loading control. b Representative BrdU-incorporation assays of U87-MG (left panel) and U87-MGshp53 cells (right panel) with overexpression of Survivin (bottom) compared to the empty vector-transduced controls (top). The overall amount of BrdU-positive fractions as well as the relative levels of BrdU+ and BrdU- cells in fractions with DNA contents of 2n, 4n, 8n and >8n are depicted. c Proliferation index of the U87-MG and U87-MGshp53 cells transduced with Survivin and empty vector controls. Depicted are mean values ± SD. **p < 0.01. d BrdU-incorporation in U87-MG and U87-MGshp53 cell fractions with DNA content >4n. Depicted are mean values ± SD. **p < 0.01. All data were collected 72 h after transduction of cells