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. 2021 Jan 29;12:704. doi: 10.1038/s41467-021-20928-8

Fig. 5. Fusion of Med17 with GOF p53 with TAD mutations regenerates transactivation and oncogenic activities.

Fig. 5

a Transactivation assay using GOF p53 inducible gene promoters and GOF p53 derivatives as indicated in the figure. Transient transcriptional assays were carried out in H1299 cells where transfections were carried out with empty vector or expression plasmid for indicated proteins. 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. The data clearly show that transactivation by GOF p53 is lost by TAD mutation and is regained by fusion of the mutated GOF p53 with Med17. Western blots are shown below to indicate the level of expression of different proteins after transfection. Asterisks indicate a p-value < 0.001. b Spheroid growth assay using H1299 p53-null lung cancer cells expressing p53-R175H, -L22Q/W23S/R175H, -R273H, -L22Q/W23S/R273H, Med17, p53-R273H + Med17, p53-22/23/R273H + Med17, p53-R273H-Med17 (fusion), p53-22/23/R273H-Med17 (fusion), or vector control. Best colonies are formed in H1299 p53-R273H cells. c Western blot analysis of cells used for assays b–h. d Quantification of the Spheroid assay in b. The number of spheroids were counted in multiple fields and plotted as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed. Colony formation ability is compromised by TAD mutations and is regained by fusion with Med17. e A tumorigenicity assay was performed using cells from spheroids in b injected subcutaneously in the flanks of SCID mice. Data are presented as the mean ± SEM (n = 4). An ANOVA was performed and **p = 0.003. The tumor-forming ability of L22Q/W23S/R273H was rescued with the fusion to Med17. f Migration assay using cells from the spheroids in b. Data are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed and *p = 0.01, **p = 0.004, and ***p < 0.0001. g Invasion assay using cells from the spheroids in b. Data are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed. h DNA replication origin firing assay using cells from the spheroids in b. Data are presented as the mean ± SEM (n = 200 DNA fibers). A two-sided Student’s t-test was performed. A cartoon is shown to depict the replication assay experimental scheme along with a representative image of a DNA fiber showing a fired origin. Migration, invasion, and origin firing abilities of L22Q/W23S/R273H were rescued with the fusion to Med17. *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.