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 α-TUBULIN serving as loading control.