Apoptosis in CD34+ dormant patient cells treated with RP2 inhibitors. (A) Ki-67/CD71 co-expression in CD34-gated primary AML cells before culture. For Ki-67 and CD71 quadrant delineation, gating was carried out strictly such that 1% of isotype control fluorescence fell into the positive quadrants. Flow cytometric dotplots of one sample and a diagram summarising mean ± standard deviation of target cell percentage in each quadrant for the seven primary samples studied are shown. (B) An example of CD71 expression in CD34+ annexin V + AML blasts. Patient cells were cultured for 16–18 hours with DRB, TG02, flavopiridol or etoposide. They were then labelled with CD71, CD45 and CD34, rinsed and additionally labelled with Annexin V. CD71 expression was determined in CD34+ early apoptotic cells using the four part gating strategy detailed in the Methods section. (i) Illustration of gating strategy showing how gates P1-P4 are applied; note especially that gates P1 and P4 are narrowed to exclude late stages of apoptosis or necrosis in order to alleviate concern that CD71 might be shed. (ii) Flow cytometric histograms showing CD71 expression in the cell subset gated on P1-P4 (MFI = mean fluorescence intensity). (C) CD71 negative cells shown as a percentage of total early apoptotic cells from the P1-P4 subset of primary samples (n = 8 for TG02 and etoposide, n = 6 for DRB and flavopiridol). As primary samples are heterogeneous, apoptosis-inducing drug concentrations were sample-specific (30-100 nM for TG02 and flavopiridol, 0.2-2 μM for etoposide, 20 μM for DRB). * The low proportions of CD71neg cells in untreated and etoposide-treated samples compared to RP2 inhibitor-treated samples were statistically significant, as detailed in the text.