C/EBPα-ER impairs self-renewal of leukemic cells. (A) Transduced AML cells were subjected to serial replating experiments in the presence of 500 nM 4-OHT (n = 6) with cells from the adherent fraction. Every 2 weeks suspension cells were removed and analyzed by flow cytometry for Venus expression. The relative number of Venus⁺ cells in suspension cultures at initial plating (week 0) are set to 1, and subsequent weeks are shown relative to initial plating. The relative number of Venus⁺ cells within the adherent human CD45⁺ fraction was analyzed as well, where the number of Venus control adherent cells was set to 1 (box on the right). A total of 10% of the adherent fraction was replated onto fresh MS5 cultures, which were maintained for another 2 weeks, followed by a second replate. The average with SEM from 6 individual AML samples is shown. Venus percentages in suspension cultures at initial plating (week 0) are set to 1, and subsequent weeks are shown relative to initial plating. (B) Representative phase-contrast microscopy images of AML cocultures in the presence of 500 nM 4-OHT on MS5 cells after the first replate at week 4 (top row; AML no. 9), or after the second replate at week 6 (bottom row; AML no. 7). CAs are circled. See “Materials and methods; Microscopy and cytospins.”