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

Figure 3.

Figure 3.

Part 1: Multiple apoptotic and necroptotic mechanisms facilitate ovarian tumor cell killing by [sorafenib + sildenafil + celecoxib]. (A) Upper: OVCAR cells were treated with vehicle; [sorafenib (2 μM) + sildenafil (2 μM)]; [celecoxib (2 μM) + sildenafil (2 μM); or all 3 drug together for 12h. Twelve h after drug treatment, cells were processed and subjected to live/dead assays in a Hermes Wiscan system. Lower: OVCAR cells were transfected with scrambled siRNA (siSCR); an siRNA to knock down AMPKα; to knock down PERK, ATF4 and CHOP. Twenty four h after transfection cells were treated with vehicle; [sorafenib (2 μM) + sildenafil (2 μM) + celecoxib (2 μM)] for 12h. Twelve h after drug treatment, cells were processed and subjected to live/dead assays in a Hermes Wiscan system. (B) Spiky cells were treated with vehicle; [sorafenib (2 μM) + sildenafil (2 μM)]; [celecoxib (2 μM) + sildenafil (2 μM)]; or all 3 drug together for 6h. Six h after drug treatment, cells were fixed in place and immuo-staining performed to detect the expression of GRP78 and the Serine 51 phosphorylation of eIF2α using a Hermes WiScan wide field microscope. (C–D) Spiky cells were treated with vehicle; [sorafenib (2 μM) + sildenafil (2 μM)]; [celecoxib (2 μM) + sildenafil (2 μM)]; or all 3 drug together for 12h. Twelve h after drug treatment, cells were fixed in place and immuo-staining performed to detect the expression of GRP78 and the Serine 51 phosphorylation of eIF2α using a Hermes WiScan wide field microscope. (E–F) OVCAR cells were treated with vehicle; [sorafenib (2 μM) + sildenafil (2 μM)]; [celecoxib (2 μM) + sildenafil (2 μM)]; or all 3 drug together for 12h. Twelve h after drug treatment, cells were fixed in place and immuo-staining performed to detect the expression of GRP78 and the Serine 51 phosphorylation of eIF2α using a Hermes WiScan wide field microscope. (G) Spiky and OVCAR cells were transfected with, as indicated: an empty vector plasmid (CMV); a scrambled siRNA (siSCR); an siRNA to knock down GRP78 expression; 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 and subjected to live/dead assays in a Hermes Wiscan system.