Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 Page 9 of 11 Figure 3 Conditional PFKFB2 over-expression in CCRF-CEM T-ALL cells has no effect on GC-induced apoptosis. CEM-PFKFB2-15A#C3, #D6 and #E8 cells (expressing PFKFB2-15A in a doxycycline-dependent manner, Figure 3A) and CEM-PFKFB2-15A#65, #66 and #95 cells (expressing PFKFB2-15B in a doxycycline-dependent manner, Figure 3B) were cultured in the absence of doxycycline and dexamethasone (open square), in the presence of 10-7 M dexamethasone (open circle) or 500 ng/ml doxycycline (closed square), and with the combination of both (closed circle) for the indicated time and subjected to apoptosis determination using flow cytometric analyses of propidium iodide-stained nuclei ("apoptosis” refers to percent of events in the SubG1 window). Since all 3 cell lines for each isoform gave similar results, the data were pooled. Shown are the mean values±SD of specific apoptosis derived from biological triplicates of the 3 cell lines (i.e., 9 measurements per time point and treatment). Figure 3C: PFKFB2-15A and -15B protein expression was analyzed in the indicated cell lines with and without 24 h doxyxcyline induction (Dox, 500 ng/ml) and in the absence and presence of dexamethasone (Dex, 10-7M) by immunoblotting using an antibody against the shared N-terminal region (top panel) or Ser466 phosphorylated PFKFB2-15A (3rd panel) with a-TUBULIN serving as loading control. children (24, 699, 724) were classified as prednisolone poor responders based on a blast count of >1000/μl on day 8, yet they also showed GC-induction of at least 1 of the variants at 1 or both time points. GC-resistant cell lines and non-leukemic individuals (whose periph- eral blood lymphocytes are considered resistant to GC-induced apoptosis) [34,35], clearly showed less GC- induction of PFKFB2. Thus, even though the correlation of GC-induction of PFKFB2 and cell death was not complete, the extended expression/regulation analyses further supported a possible role of PFKFB2 in GC-induced apoptosis, particularly in T-ALL cells. We therefore performed conditional over-expression of both PFKFB2 splice variants in a GC-sensitive T-ALL model system, but these experiments showed no detect- able effect of PFKFB2-15A or -15B on cell death, nor did their over-expression change sensitivity to, or kinetics of, GC-induced apoptosis. While these data clearly showed that induction of neither of the 2 splice variants suffices to elicit the anti-leukemic GC effects,