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. 2015 Sep 29;16(11):1660–1670. doi: 10.1080/15384047.2015.1099769

Figure 4.

Figure 4.

Part 2: Multiple apoptotic and necroptotic mechanisms facilitate ovarian tumor cell killing by [sorafenib + sildenafil + celecoxib]. (A) Spiky and OVCAR cells were transfected with an empty vector plasmid (CMV) or a plasmid to express a dominant negative eIF2α S51A protein. Twenty four h after transfection cells were treated with vehicle or with [sorafenib (2 μM) + sildenafil (2 μM) + celecoxib (2 μM)]. Twelve h after drug treatment, cells were processed were fixed in place and immuo-staining performed to detect the expression of: MCL-1, BCL-XL, c-FLIP-s and cleaved caspase 3, using a Hermes WiScan wide field microscope. (B) OVCAR cells were transfected with, as indicated: an empty vector plasmid (CMV); a scrambled siRNA (siSCR); an siRNA to knock down RIP-1 expression; and plasmids to express c-FLIP-s, BCL-XL and dominant negative caspase 9 proteins. Twenty four h after transfection cells were treated with vehicle or with [sorafenib (2 μM) + sildenafil (2 μM) + celecoxib (2 μM)]. Twelve h after drug treatment, cells were processed and subjected to live/dead assays in a Hermes Wiscan system. (C) OVCAR cells were transfected with, as indicated: a scrambled siRNA (siSCR); and siRNA molecules to knock down ATG5 or Beclin1 expression. Twenty four h after transfection cells were treated with vehicle or with [sorafenib (2 μM) + sildenafil (2 μM) + celecoxib (2 μM)]. Twelve h after drug treatment, cells were processed and subjected to live/dead assays in a Hermes Wiscan system. (D) OVCAR and Spiky cells were transfected with, as indicated: a scrambled siRNA (siSCR); and siRNA molecules to knock down BID or [caspase 2 + caspase 4] expression. Twenty four h after transfection cells were treated with vehicle or with [sorafenib (2 μM) + sildenafil (2 μM) + celecoxib (2 μM)]. Twelve h after drug treatment, cells were processed and subjected to live/dead assays in a Hermes Wiscan system.