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. 2017 Jan 9;8(8):13464–13475. doi: 10.18632/oncotarget.14562

Figure 5. Sildenafil-induced PKG signaling plays a greater role in enhancing [pemetrexed + sorafenib] toxicity than nitric oxide synthase signaling.

Figure 5

(A) H460 cells were transfected with a scrambled siRNA (siSCR) or molecules to knock down the expression of IRE1 or XBP1. In parallel, cells were transfected with an empty vector plasmid (CMV) or with plasmids to express: HSP90; GRP78; HSP70; HSP27 or in the indicated combinations. Twenty-four h after transfection cells were treated with vehicle control or [pemetrexed (1.0 μM) + sildenafil (2.0 μM) + sorafenib (2.0 μM)] for 12 h. Floating cells were then cytospun onto the 96 well plate and cell viability determined. The percentage cell death under each transfection and treatment condition was calculated and values with a statistical significance lower than the corresponding value CMV cells are in red; those whose value is greater than in CMV cells are green (*p < 0.05 less than corresponding CMV cells; #p < 0.05 greater than corresponding value in siSCR cells). (B) NSCLC cells were transfected with a scrambled siRNA control (siSCR) or transfected to knock down the expression of PKGI and PKGII. Twenty-four h after transfection cells were treated with vehicle control or [pemetrexed (1.0 μM) + sorafenib (2.0 μM) + sildenafil (2.0 μM)] in combination for 24 h. Thirty minutes prior to drug treatment, cells were treated with vehicle control or with the nitric oxide synthase inhibitor L-NAME (1 μM). Floating cells were cytospun onto the 96 well plate and viability determined using a live/dead viability stain where green cells are viable and yellow/red cells are dead (n = 3 +/−SEM) *p < 0.05 less than corresponding value in siSCR cells; #p < 0.05 less than corresponding value in siSCR + L-NAME cells. (C) NSCLC cells were transfected with a scrambled control siRNA (siSCR) or with siRNA molecules to knock down the expression of: iNOS and eNOS; PKGI and PKGII, as indicated. Twenty-four h after transfection cells were treated with vehicle control or [pemetrexed (1.0 μM) + sorafenib (2.0 μM) + sildenafil (2 μM)] in combination for 24 h. Floating cells were cytospun onto the 96 well plate and viability determined using a live/dead viability stain where green cells are viable and yellow/red cells are dead (n = 3 +/−SEM) *p < 0.05 less than corresponding value in siSCR cells; #p < 0.05 less than values in individual NOS knock down cells.