Figure 6. Further polyploidization of EVI1-induced tetraploid cells is inhibited by p53. (A) Parental U2OS cells and U2OS cells conditionally overexpressing EVI1-HA were transfected with siRNAs targeting luciferase (siluc) or p53 (sip53), respectively. Twenty-four hours later, transgene expression was induced where indicated (+TET), and cells were harvested 72 h thereafter, followed by immunoblotting for the indicated proteins. (B) U2OS cells retrovirally transduced to conditionally overexpress EVI1 were treated as in (A), followed by immunoblotting for the indicated proteins. (C) U2OS cells conditionally overexpressing EVI1-HA were transfected with siRNAs targeting luciferase (siluc) or p53 (sip53), respectively. Twenty-four hours later, transgene expression was induced where indicated (+TET), and cells were harvested 72 h thereafter, followed by immunostaining for HA (red) and γ-tubulin (green). DNA was counterstained with DAPI (blue). Scale bars represent 10 µm. Insets show centrosomes at higher magnification. (D) U2OS cells conditionally overexpressing EVI1-HA were transfected with siRNAs targeting luciferase (siluc) or p53 (sip53), respectively. Twenty-four hours later, transgene expression was induced where indicated (+TET), and cells were harvested 72 h thereafter, followed by flow-cytometric analysis of DNA content. This included gating in cells of interest (blue) by excluding doublets, followed by defining regions containing cells with 8N DNA (red), corresponding to the given percentages, or more than 4N DNA (not shown). The complete data are listed in Table S1. (E) Flow-cytometric analysis of polyploidization was performed exactly as described in (D), except that U2OS cells retrovirally transduced to conditionally overexpress EVI1 were used. The complete data are listed in Table S1.