AML1-ETO suppressed separase and PU.1 expression. (A) Northern blotting for separase (top) with days 0, 1, and 2 of tetracycline-removed (−Tet) U937T-AML1-ETO (left) and parental U937T (right) cells was performed. The ethidium bromide staining of the 18S rRNA shows the relative loading of RNA. (B) Separase expression was examined by RT-qPCR with days 0, 1, and 2 of tetracycline-removed U937T-AML1-ETO (left) and U937T (right) cells. The relative separase mRNA level compared with day 0, which is set to 1, is shown. The results are presented as the mean value of 3 independent experiments + standard deviation. Statistically significant P value (t test) is indicated only for U923T-AML1-ETO. (C) Immunoblotting with protein extracts from U937T-AML1-ETO (left) or U937T cells (right) was performed using anti-separase antibody. Both full-length (220 kDa) and cleaved (150 kDa) forms of separase were detected (). Tubulin blots are shown as loading controls. (D) Northern blotting for PU.1 (top) with days 0, 1, and 2 of tetracycline-removed (−Tet) U937T-AML1-ETO (left) and parental U937T (right) cells was performed. The ethidium bromide stainings of the 18S rRNA are also shown (bottom).