Fig 2: Human p53-R273H TAD defects reduce oncogenicity of H1299 cells.a Spheroid growth assay with H1299 p53-null lung cancer cells expressing p53-R273H, -L22Q/W23S/R273H, or vector control. Representative images are shown from each cohort. b Quantification of spheroid assay. The number of colonies were counted across multiple fields and plotted as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed, **p = 0.0093. The most colonies are formed in H1299 p53-R273H cells. c A tumorigenicity assay was performed using cells from the spheroids formed in a injected subcutaneously in the flanks of NOD SCID mice. Data are presented as the mean ± SEM (n = 4). A two-sided Student’s t-test was performed comparing the vector cell lines, **p = 0.0369 and ***p = 0.00017. Vector control and L22Q/W23S/R273H grew much slower compared to R273H. d Western blot showing levels of p53 in cell clones. e Invasion assay performed using cells from the spheroids in a. Data are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed, **p = 0.0022 and ***p = 6.18E–6. Sphere formation, tumorigenicity, and invasion are all affected by mutation at 22/23.

Fig 3: GOF p53 interacts with Med17 and amino acid acids at mouse p53 L25, W26 (L22, W23 in human) are important for this interaction.a Immunoprecipitation experiment showing complex formation between murine p53-R172H and the Mediator protein 17 (Med17) in solution. Nuclear extracts were made from murine lung cells expressing p53-R172H (generated by adeno Cre as described in the text), p53-L25Q/W26S/R172H, and control, and immunoprecipitated by Med17 antibody and probed with anti-p53 antibody. b Immunoprecipitation experiment showing complex formation between p53-R273H with Med17 in solution. Nuclear extracts were prepared from H1299 cells stably transfected with empty vector, expressing p53-R273H and -L22Q/W23S/R273H, and immunoprecipitated by Med17 antibody and probed with anti-p53 antibody. c Immunoprecipitation experiment showing complex formation between p53-R175H and Med17 in solution. Nuclear extracts were prepared from H1299 cells stably transfected with empty vector, expressing p53-R175H and -L22Q/W23S/R175H, and immunoprecipitated by Med17 antibody and probed with anti-p53 antibody. d Reverse immunoprecipitation experiment showing complex formation between murine p53-R172H and the Mediator protein 17 (Med17) in solution. Whole cell extracts were made from murine lung cells expressing p53-R172H (generated by adeno Cre as described in the text), p53-L25Q/W26S/R172H, and control after transfection with Flag-Med17, and immunoprecipitated by p53 antibody and probed with anti-Flag antibody. e Reverse immunoprecipitation experiment showing complex formation between p53-R273H and the Mediator protein 17 (Med17) in solution. Whole cell extracts were made from H1299 cells stably transfected with empty vector, expressing p53-R273H and -L22Q/W23S/R273H after transfection with Flag-Med17, and immunoprecipitated by p53 antibody and probed with anti-Flag antibody. f Reverse immunoprecipitation experiment showing complex formation between p53-R175H and the Mediator protein 17 (Med17) in solution. Whole cell extracts were made from H1299 cells stably transfected with empty vector, expressing p53-R175H and -L22Q/W23S/R175H after transfection with Flag-Med17, and immunoprecipitated by p53 antibody and probed with anti-Flag antibody. g ChIP-re-ChIP experiment showing complex formation on mutant p53 inducible gene promoters (cyclin A2 and chk1) between mutant p53 and Med17. ChIP-re-ChIP was carried out as described using murine lung cells described in a and H1299 cells described in b. The first immunoprecipitation was carried out with p53 antibody, while the second one was done using a Med17 antibody. Presence of promoter sequences was detected by QPCR using promoter specific primers. Data are normalized with input DNA values for each respective promoter and is presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed; Mouse CCNA2 *p = 0.009, **p = 0.003, Mouse Chk1 **p = 0.007, ***p = 3.12E–5; Human CCNA2 **p = 0.008, ***p = 0.0002, Human Chk1 *p = 0.01, **p = 0.005. h ChIP-re-ChIP experiment showing complex formation on wild-type (WT) p53 inducible gene promoter p21 between WT p53 and Med17. ChIP-re-ChIP was carried out as described using H460 (WT p53) and HCT116 (p53+/+). The first immunoprecipitation was carried out with p53 antibody, while the second one was done using a Med17 antibody. Presence of promoter sequences was detected by QPCR using promoter specific primers. Data are normalized with input DNA values and is presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed; H460 DMSO *p = 0.001, ***p = 0.0007; H460 Dox *p = 9.6E–4, **p = 1.05E–5, ***p = 2.29E–4; HCT116+/+ DMSO *p = 0.02, **p = 0.006, ***p = 0.009, HCT116+/+ Dox *p = 0.004, **p = 6.3E–5, ***p = 2.2E–4. Assay was performed with or without Dox treatment. NS no statistically significant difference from control.

Fig 4: TAD mutations affect the state of phosphorylation of mutant p53.Western blot analysis of equal amounts of extracts from a H1299 cells expressing p53-R273H, -L22Q/W23S/R273H, and vector control; b H1299 cells expressing p53-R175H, -L22Q/W23S/R175H, and vector control; and c murine lung cells expressing p53-R172H, -L22Q/W23S/R172H, and p53–/– control were developed with antibodies against different phosphorylation sites on p53. The data presented show that phosphorylation at amino acid position 20 went down significantly. d Transactivation assays of mutant p53 and its derivatives by Ala substitution mutations at positions of phosphorylation using a mutant p53 inducible gene promoter. Transient transcriptional assays were carried out in H1299 cells after transfection of NF-?B2-luc and indicated expression plasmid or empty vector. Cells were harvested after 24 h of transfection. Firefly luciferase readings were plotted as relative light units (RLU). Data are presented as the mean ± SEM (n = 3, performed twice). A two-sided Student’s t-test was performed. Ser20Ala substitution reduced the transactivation ability the greatest. Substitution of S20 to Glu or Asp did not regain transactivation ability either. e Western blot analysis showing level of different p53 mutants after transfection. *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.

Fig 5: Transcriptome analyses of NSF-HA-treated IMR90 cells.a Genes differentially expressed in IMR90 cells upon treatment with cNSF-HA or fNSF-HA (HA polymer length-dependent genes). Cells were pre-incubated for 6 h with 20 µg/ml cNSF-HA, fNSF-HA, or PBS and then HA was removed and cells were exposed to 200 µM tBHP for 1 h. Cells were collected 6 h after starting the tBHP-treatment. b Enrichment of GO Term, Reactome pathway, and transcription factor protein-protein interactions (PPIs) in the HA polymer length-dependent genes. c, d Enrichment of transcription factor binding sites (TFBS) in the HA polymer length-dependent genes. White bars represents the enrichment of TFBS in all HA polymer length-dependent genes. Gray bars represents the enrichment of TFBS in HA polymer length-dependent genes that are involved in the transcriptional regulation by p53 or encoding p53-interacting proteins. Black bars represents the enrichment of TFBS in HA polymer length-dependent p53 target genes. e Differential expression levels of the HA polymer length-dependent genes (defined in the presence of tBHP) in IMR90 cells incubated for 6 h with 20 µg/ml cNSF-HA, fNSF-HA, or PBS. f The violin plot represents the distribution of p-values of a two-tailed t-test comparing the gene expression levels of the HA polymer length-dependent genes and HA-polymer length-independent genes (defined in the presence of tBHP) between cNSF-HA- and fNSF-HA-incubated IMR90 cells. Cells were incubated for 6 h with 20 µg/ml cNSF-HA or fNSF-HA without further tBHP-treatment. g Differential expression levels of the HA polymer length-dependent genes (defined in the presence of tBHP) in control and CD44-overexpressing IMR90 cells incubated for 6 h with 20 µg/ml cNSF-HA or PBS.