Figure 8. Death receptor signaling and autophagosome formation are essential for neratinib to enhance [regorafenib + sildenafil] lethality.

A. CT26 cells were transfected with a scrambled control siRNA (siSCR) or were transfected to knock down the expression of the indicated proteins (see Figure 7 for knock down images). Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 12h. Cells were then isolated, and viability determined by trypan blue exclusion assay (n = 3 +/− SD). * p < 0.05 greater than reg/sil value in siSCR cells; ** p > 0.05 greater than reg/sil value in siSCR cells. B. CT26 cells were transfected with an empty vector control plasmid (CMV) or were transfected with plasmids to express the indicated proteins: c-FLIP-s; BCL-XL; dominant negative caspase 9 (see Figure 7 for expression images). Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 12h. Cells were then isolated, and viability determined by trypan blue exclusion assay (n = 3 +/− SD). * p < 0.05 greater than reg/sil value in siSCR cells; ** p > 0.05 greater than reg/sil value in siSCR cells. C. PANC1, PAN02 and CT26 cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 3h. Cells were fixed in place without membrane permeabilization. Immunofluorescent staining performed to determine the total plasma membrane levels of CD95. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD); # p < 0.05 greater than values in [R+S] treatment.