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. 2018 Nov 2;20(5):597–607. doi: 10.1080/15384047.2018.1538616

Figure 2.

Figure 2.

[Pemetrexed + fingolimod] treatment inactivates mTOR, activates the AMPK and ULK1 and simultaneously induces autophagosome and autolysosome formation. A. NSCLC cells were treated with vehicle control or with [pemetrexed (500 nM) + fingolimod (200 nM)] for 6h. After six h, cells were fixed in place and immunostaining performed to detect the expression and phosphorylation of the indicated proteins. Staining densities of phospho-/total-protein levels are determined in 40 cells in triplicate using software integral in the Hermes WiScan microscope (n = 120 cells ± SEM). # p < 0.05 greater than vehicle control value; * p < 0.05 less than vehicle control value. B. NSCLC cells were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection, cells were treated with vehicle control or with [pemetrexed (500 nM) + fingolimod (200 nM)] for 3h, 6h or 12h. At each time point the mean number of intense staining GFP+ and RFP+ vesicles in the cells was determined (at least 40 cells per condition were counted) (n = 120 cells ± SEM). # p < 0.05 greater than vehicle control value.