Figure 5.

Changes in Mcl-1 protein level do not account for sensitisation of neuroblastoma cells to ABT-737 in hypoxia. A. No changes in levels of Bcl-2, Bcl-XL, Mcl-1, noxa or Bid were seen in 4 neuroblastoma cell lines after 48 hr exposure to hypoxia. Actin is shown as a loading control. Blots are representative of 3 independent experiments. B. No changes in protein levels of Bcl-2, Mcl-1, Bid and Noxa were seen over a 64 hr timecourse after transfection of SH-EP1 cells with either non-target or HIF-1α siRNA, despite the dramatic loss of sensitisation to ABT-737 in hypoxia following transfection with HIF-1α siRNA. Actin is shown as a loading control. Blots are representative of 3 independent experiments. C. Reduction in protein expression of Mcl-1 in SH-EP1 cells over a 64 hr timecourse in SH-EP1 clone stably expressing short hairpin RNA against Mcl-1 in comparison with non-target short hairpin. Actin is shown as a loading control. Blots are representative of 3 independent experiments. D. Reduction in protein level of Mcl-1 increases ABT-737-induced apoptosis in normoxia as shown by a significant increase in % of annexin V/7-AAD positive cells 24 hrs after treatment with either 1 or 5μM ABT-737 (* p<0.02 at 1μM and **p<0.05 at 5μM by Student t-test). No increase in ABT-737-induced apoptosis is seen in hypoxia. E. Reduction in protein level of Mcl-1 sensitises SH-EP 1 cells to ABT-737 in normoxia, but not in hypoxia in SRB assay. Compare the dose response curve for cell expressing non-target hairpin (open diamonds) with cells stably expressing shRNA which targets Mcl-1 (closed triangles).