Figure 8. EVI1 overexpression interferes with proliferation and induces a negative feedback loop. (A) Of parental U2OS cells, uninduced (-TET) and induced (+TET) U2OS cells conditionally overexpressing EVI1-HA, a measure of 200,000 cells per dish was seeded. At the indicated time points, cells were harvested and counted again to determine proliferation rates. Results are given as mean absolute numbers of cells from two experiments. ***, this difference is highly significant (p = 0.00035). **, these differences are significant (p < 0.009). (B) In U2OS cells conditionally overexpressing EVI1-HA, transgene expression was induced for the indicated times, and tetracycline-containing medium was renewed every 48 h. Using 70 µg of protein extract per lane from induced cells (+TET) or uninduced controls (-TET), immunoblots for HA and the loading control actin were prepared.