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. 2016 Nov 26;8(1):1449–1468. doi: 10.18632/oncotarget.13640

Figure 11. PKG-dependent basal and stimulated chaperone phosphorylation is required for the inhibition of HSP90 and HSP70 by [pemetrexed + sildenafil].

Figure 11

A. H460 cells were transfected with a plasmid to express FLAG-HSP90 or with a plasmid to express HA-HSP70. Twenty-four h after transfection the cells were treated with vehicle control, pemetrexed (1.0 μM), sildenafil (2 μM)] or the drugs in combination for one hour. Cells were then lysed and HSP90 and HSP70 immuno-precipitated using their FLAG and HA tags. The ATPase activity of each chaperone was determined as described in the Methods (n = 3 +/− SEM) * p < 0.05 less than vehicle control; ¶p < 0.05 less than pemetrexed single agent value. B-D. NSCLC cells were transfected with a scrambled control siRNA (siSCR) or with siRNA molecules to knock down the expression of: AMPKα; iNOS and eNOS; PKGI and PKGII, as indicated. In parallel, cells were transfected with a plasmid to express FLAG-HSP90 or with a plasmid to express HA-HSP70. Twenty-four h after transfection the cells were treated with vehicle control or [pemetrexed (1.0 μM) + sildenafil (2 μM)] in combination for one hour. Cells were then lysed and HSP90 and HSP70 immuno-precipitated using their FLAG and HA tags. The ATPase activity of each chaperone was determined as described in the Methods (n = 3 +/− SEM) * p < 0.05 less than vehicle control; ¶p < 0.05 less than pemetrexed single agent value; # p < 0.05 greater than corresponding value in siSCR transfected cells.