Figure 2. Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells.

Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.