Figure 7
The phosphorylation of Bad and CREB in cAMP- and DNR-exposed leukemic cells. (a) NB4 cells were treated with DNR (5 μM) for 3 h alone or in combination with PGE2/IBMX (2 μM/0.15 mM), and extracts analyzed by immunoblotting for Bad, P-Ser118Bad or P-Ser75Bad, with actin as loading control. The migration corresponding to proteins of Mw 20 and 18.5 kDa is based on interpolation of the migration of relevant standard proteins. (b) Blasts isolated from a patient diagnosed with APL (APL3, Supplementary Table S1) were treated in vitro for 3 h with PGE2/IBMX (2 μM/0.3 mM). Cell extracts were analyzed by immunoblotting for Bad and P-Ser118Bad. (c) HL60 cells were treated for 3 or 6 h with DNR (5 μM) alone or in combination with PGE2/IBMX (2 μM/0.15 mM), and cell extracts were analyzed by immunoblotting for Bad and P-Ser118Bad. (d) Mv4-11 cells were treated for 5 h with DNR (0.4 μM) alone or in combination with PGE2/IBMX (2 μM/0.2 mM), and cell extracts were analyzed by immunoblotting for Bad and P-Ser118Bad. (e) The NB4 cell death was scored by microscopy after incubation with various concentrations of the BH3 mimetic HA14-1, and DNR (3 μM) and PGE2/IBMX (2 μM/0.15 mM). For the left subpanel, the cells were incubated for 3.5 h with 10 μM HA14-1 either alone or during the last 3 h with DNR, PGE2/IBMX or both. For the right hand subpanel, the incubation time with HA14-1 (4 or 6 μM) was 9.5 h, during the last 9 h with DNR, PGE2/IBMX or both. Data are shown as mean±S.E.M., n=3–6. Significance by Student t-test: **P<0.01, *P<0.05. (f) Phospho-protein analysis by flow cytometry for CREB, Erk1/2, Akt, STAT1, STAT5 and STAT3 (see Materials and Methods for detail of phospho-residue detected). NB4 cells were incubated for 1 or 3 h with DNR (5 μM), the PKA agonist N6-Bz-cAMP (0.4 mM), the Epac agonist 8-CPT-2′-Me-cAMP (0.5 mM) or the cAMP-elevating agents PGE2/IBMX (2 μM/0.1 mM). The phospho-epitope labeling intensity is shown as histograms (left subpanels) and heat maps (right subpanels). The control sample was used as a baseline for the heat maps. See Materials and Methods for further experimental details. (g) Phospho-protein analysis by flow cytometry for CREB in blasts isolated from three patients (APL3, AML3 and AML4; Supplementary Table S1). Blasts were treated in vitro for 3 h with PGE2/IBMX (2 μM/0.3 mM) or N6-Bz-cAMP (0.6 mM) before fixation and analysis by flow cytometry. The phospho-epitope labeling intensity is shown as histograms (left subpanels) and heat maps (right subpanels). The control sample was used as a baseline for the heat maps
