Skip to main content
. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Mol Cancer Ther. 2014 Aug 7;13(10):2384–2398. doi: 10.1158/1535-7163.MCT-14-0172

Figure 5. Knock down of IRE1 enhances OSU-03012 and (OSU-03012 + sildenafil) toxicity through caspase dependent pathways.<.

Figure 5

br>(A) GBM6 cells were transfected with either a scrambled siRNA (siSCR) or with an siRNA to knock down expression of IRE1. Thirty six h after transfection cells were pre-treated with the pan-caspase inhibitor zVAD (50 μM) then treated with vehicle (VEH, DMSO) or OSU-03012 (1.0 μM). Twenty four h after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3 +/− SEM) * p < 0.05 greater than corresponding value in siSCR treatment; # p < 0.05 less than (siIRE1 + OSU-03012) value. Inset blots: GBM6 cells were transfected with either a scrambled siRNA (siSCR) or with an siRNA to knock down expression of IRE1. Thirty six h after transfection cells were treated with vehicle (VEH, DMSO) or OSU-03012 (1.0 μM). Six h after treatment cells were lysed and immunoblotting performed to determine the expression of MCL-1 and BCL-XL and the phosphorylation of PERK. (B) GBM6 cells were transfected with either a scrambled siRNA (siSCR) or with an siRNA to knock down expression of XBP1. Thirty six h after transfection cells were pre-treated with the pan-caspase inhibitor zVAD (50 μM) then treated with vehicle (VEH, DMSO) or OSU-03012 (1.0 μM). Twenty four h after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3 +/− SEM) * p < 0.05 greater than corresponding value in siSCR treatment; # p < 0.05 less than (siXBP1 + OSU-03012) value. (C) GBM6 cells were transfected with either a scrambled siRNA (siSCR) or with an siRNA to knock down expression of IRE1. Thirty six h after transfection cells were pre-treated with the caspase 9 inhibitor LEHD (50 μM) then treated with vehicle (VEH, DMSO) or OSU-03012 (1.0 μM). Twenty four h after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3 +/− SEM) * p < 0.05 greater than corresponding value in siSCR treatment; # p < 0.05 less than (siIRE1 + OSU-03012) value. (D) GBM6 and GBM12 cells were infected at 50 m.o.i. with either an empty vector adenovirus (CMV) or viruses to express dominant negative caspase 9, BCL-XL or the caspase 8 inhibitor c-FLIP-s. Twenty four h after infection cells were treated with vehicle (DMSO) or with OSU-03012 (1.0 μM) and sildenafil (2 μM) combined. Twenty four h after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3 +/− SEM) # p < 0.05 less than corresponding value in CMV infected cells; ## p < 0.05 less than value in BCL-XL infected cells. Inset Panel: Cells were treated with vehicle (DMSO), OSU-03012 (1.0 μM), sildenafil (2 μM) or the drugs combined. Six h after treatment cells were lysed and immunoblotting performed to determine the expression of MCL-1, BCL-XL and c-FLIP-s. (E) GBM6 and GBM12 cells were transfected with either a scrambled siRNA (siSCR) or with siRNA molecules to knock down expression of CD95, FADD or RIP-1. Thirty six h after transfection cells were treated with vehicle (DMSO) or with OSU-03012 (1.0 μM) and sildenafil (2 μM) combined. As a positive control one set of siSCR transfected cells were treated with an agonistic azide free anti-CD95 antibody (2 μg) (the use of a control IgG was also performed but is not shown for clarity). Twenty four h after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3 +/− SEM) # p < 0.05 less than corresponding value in siSCR transfected cells. (F) GBM6 cells in 96 well plates were pre-treated with vehicle or L-NAME (1 μM). Cells were then treated with OSU-03012 (OSU 1.0 μM) and/or sildenafil (SIL, 2.0 μM). Six h after treatment cells were fixed to the plate and immunohistochemistry performed to determine the plasma membrane levels of CD95. The intensity of CD95 immunostaining was determined using a Hermes Wiscan instrument with associated Wisoft data analysis package (n = 3 +/− SEM). * p < 0.05 value greater than celecoxib treatment alone; # p < 0.05 less than corresponding value in vehicle treated cells.