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. 2010 Oct 15;96(1):69–77. doi: 10.3324/haematol.2010.026997

Figure 3.

Figure 3.

Synergistic cell death was caspase-dependent and p53-independent. (A) NALM6 and REH cells were either untreated, treated with 16 μM RAD001, exposed to indicated doses of ionizing radiation or both RAD001 and ionizing radiation for 24 h. Cells were labeled for cleaved caspase-3 and analyzed by flow cytometry. Representative graphs from duplicate determinations are shown. (B) REH cells were treated with 16 μM RAD001 and 2.5 Gy of ionizing radiation in the presence or absence of the pan-caspase inhibitor ZVAD-FMK (20 μM). Cells were labeled and data shown as for (A). (C) NALM6 cells were either untreated or exposed to 1 Gy of ionizing radiation (IR) or the indicated chemotherapeutic agents, 16 μM RAD001 or the combination of RAD001 and radiation or chemotherapeutic agents in the absence or presence of 20 μM ZVAD-FMK for 24 h. Chemotherapeutic agents were used at the following concentrations: 10 μM doxorubicin (Dox) or 100 nM etoposide (Etop). Cells negative for annexin V and 7-AAD staining by flow cytometry were considered viable. The mean±SD of triplicate determinations are shown. † Indicates a significant synergistic interaction between RAD001 and the cytotoxic agent (P<0.05). *Indicates a significant reversal of the synergistic killing (P<0.05). (D) NALM6, REH and LK63 cells were untreated or exposed to 10 nM vincristine, in the absence or presence of the p53 inhibitor pifithrin (25 μM) for 12 h. Cells were analyzed by flow cytometry, and those negative for annexin V and 7-AAD staining were considered viable. The mean±SD of triplicate determinations is shown. (E) NALM6 cells were transduced with lentiviral constructs containing either luciferase shRNA-GFP (Control) or p53 shRNA-GFP and treated with vehicle or 5 nM vincristine. Cells were labeled for p53 and analyzed by flow cytometry using GFP to identify successfully transduced cells. The mean±SD of duplicate determinations is shown. *Indicates a significant suppression of p53 induction in response to vincristine. (F) NALM6 cells were transduced with p53 shRNA-GFP or luciferase shRNA-GFP and treated with 5 nM vincristine or vehicle for 12 h. Cells were labeled for cleaved caspase-3 and analyzed by flow cytometry, the percentage of cells lacking cleaved caspase-3 is shown. §P<0.05 compared to the equivalently treated luciferase shRNA-GFP cells.