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

Fig. 8. PLK3 controls GOF p53’s transactivation and binding to its inducible gene promoters.

Fig. 8

a Transient transactivation assay of H1299 cells transfected with the p53-R273H (or empty vector) along with siRNA against PLK3 (or scrambled control) and the cyclin A promoter, Chk1 promoter, or the TERT promoter-luciferase construct. Transient transcriptional assays were carried out in H1299 cells. Cells were harvested after 24 h of transfection. Firefly luciferase readings were plotted as relative light units (RLU) and is presented as the mean ± SEM (n = 3, performed twice). A two-sided Student’s t-test was performed and CCNA2 *p = 0.01, ***p = 6.5E–7; Chk1 ***p < 2.5E–6; TERT *p = 0.01. b H1975 PLK3 KO cells were used to determine the ability of GOF p53 to upregulate its activated genes. Transcript levels of PLK3, Chk1, CCNA2, and EIF3C were measured by QPCR. Data are normalized with GAPDH and are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed and PLK3 **p = 0.002; Chk1 **p = 0.001; CCNA2 *p = 0.03; EIF3C *p = 0.02. c Co-immunoprecipitation of mutant p53 and PLK3 in murine and human cells. Mouse R172H and human R273H were able to interact with PLK3 but their transactivation-deficient counterparts, L25Q/W26S/R172H and L22Q/W23S/R273H were not. d PLK3 knockout (KO) cells were used to determine whether the interaction between mutant p53 and Med17 has any requirement for PLK3. Co-immunoprecipitation in H1975 PLK3 KO cells versus control showed that the interaction between mutant p53 and Med17 is lost when PLK3 is knocked out. e The ability of mutant p53 to bind to its inducible promoters was determined by performing a ChIP assay. H1299 cells expressing p53-R273H (or containing empty vector) were transfected with siRNA against PLK3 (or scrambled control) and p53 ChIP was performed. 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 and *p = 0.02. f ChIP-reChIP assays were performed to determine if the complex between PLK3 (and/or Med17) and mutant p53 are present on mutant p53’s inducible promoters using H1299 cells stably expressing the p53-R273H or -L22Q/W23S/R273H (or containing empty vector). The first immunoprecipitation was carried out with Med17 or PLK3 antibodies while the second IP was done using a p53 antibody (or IgG control). The presence of promoter sequences was detected by QPCR using promoter specific primers. Data are normalized with input DNA values for each respective promoter and are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed and *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.