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

Figure 6. AKT / mTOR signaling is protective against [pemetrexed + sildenafil].

Figure 6

A. NSCLC cells were transfected with a scrambled siRNA control (siSCR) or transfected to knock down the expression of ATM or of AMPKα. After 24h, cells were fixed in place and immuno-fluorescence staining performed to determine the phosphorylationof AKT T308 and AKT S473. B. NSCLC cells were treated with vehicle control or [pemetrexed (1.0 μM) + sildenafil (2 μM)] and/or Temsirolimus (0.5 μM) in combination as indicated for 24h. 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 the corresponding value in siSCR cells. C. NSCLC cells were transfected with a scrambled siRNA or were transfected to knock down the expression of mTOR. After 24h, cells were treated with vehicle control or with [pemetrexed 1.0 μM + sildenafil 2.0 μM]. After a further 24h 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 the corresponding value in siSCR cells. D. NSCLC cells were transfected with a scrambled siRNA control or were transfected to knock down expression of ATM. After 24h, cells were treated with vehicle control or with [Temsirolimus 0.5 μM + sildenafil 2.0 μM]. After a further 24h 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 the corresponding value in siSCR cells. E. NSCLC cells were transfected with an empty vector plasmid or a scrambled siRNA control or were transfected to express dominant negative AKT or knock down expression of mTOR and/or to knock down the expression of ATM or of AMPKα. After 24h, 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 the corresponding value in siSCR cells.