Figure 2. [Pemetrexed + sorafenib + sildenafil] treatment inactivates cyto-protective STAT3, STAT5 and AKT whilst reducing the expression of cyto-protective proteins MCL-1, BCL-XL and Thioredoxin.

(A) Parental clones of H1975 cells and afatinib resistant clones of H1975 cells were treated for 6 h with vehicle control or with [pemetrexed (1.0 μM), sildenafil (2.0 μM), sorafenib (2.0 μM)] in combination as indicated. Cells were fixed in place and immuno-fluorescence staining performed to determine the phosphorylation and expression of the indicated proteins *p < 0.05 less than corresponding intensity in vehicle control cells; ^#p < 0.05 greater than corresponding intensity in vehicle control cells. (B) Parental clones of H1975 cells and afatinib resistant clones of H1975 cells were treated for 6 h with vehicle control or with [pemetrexed (1.0 μM), sildenafil (2.0 μM), sorafenib (2.0 μM)] in combination as indicated. Cells were fixed in place and immuno-fluorescence staining performed to determine the phosphorylation and expression of the indicated proteins. *p < 0.05 less than the mean of the corresponding values in wild type control cells; ^#p < 0.05 less than the mean of the corresponding vehicle treated values in wild type control cells. (C) A549 and H1975 NSCLC cells were treated for 2 h–12 h as indicated with vehicle control or with pemetrexed (1.0 μM), sildenafil (2.0 μM), sorafenib (2.0 μM), alone or in combination as indicated. Cells were fixed in place and immuno-fluorescence staining performed to determine the phosphorylation and expression of the indicated proteins.