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. 2017 Oct 9;8(52):90262–90277. doi: 10.18632/oncotarget.21660

Figure 3. Neratinib lethality requires autophagosome formation.

Figure 3

(A) Afatinib resistant clones were treated with vehicle control or with neratinib (0.5 μM). After 6h cells were fixed in place and immunofluorescent staining performed to detect the phosphorylation of mTOR S2448 (mTORC1) and mTOR S2481 (mTORC2). (n = 3 +/-SEM) * p < 0.05 lower staining intensity than in vehicle control treated clones. (B) Afatinib resistant clones were treated with vehicle control or with sodium valproate (250 μM). After 6h cells were fixed in place and immunofluorescent staining performed to detect the phosphorylation of mTOR S2448 (mTORC1) and mTOR S2481 (mTORC2). (n = 3 +/-SEM) * p < 0.05 lower staining intensity than in vehicle control treated clones; # p < 0.05 higher staining intensity than in vehicle control treated clones. (C) Afatinib resistant clones were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection cells were treated with vehicle control or with [neratinib (0.5 μM) + valproate (250 μM)] for 6h and for 12h. The number of intense staining GFP+ and RFP+ foci was determined from 40 cells per condition. (n = 3 +/-SEM) # p < 0.05 greater than vehicle control. (D) An afatinib resistant clone was treated with vehicle control or with neratinib for 24h. Cells were stained with live/dead reagent and images at 10X magnification obtained. Arrows indicate the presence of large vesicle structures in the cells. (E) Afatinib resistant clones were transfected with a scrambled control siRNA or with an siRNA to knock down Beclin1. Twenty-four h later, cells were treated with vehicle control or with [neratinib (0.5 μM) + valproate (250 μM)]. After an additional 24h cells were treated with live/dead reagent and the percentage cell death under each condition determined. Data are the mean death from the 5 clones. (n = 3 +/-SEM) * p < 0.05 less killing compared to siSCR cells. The representative inset panel shows total cell numbers. (F) Afatinib resistant clones were transfected with an empty vector plasmid or a plasmid to express an activated form of mTOR. Twenty-four h later, cells were treated with vehicle control or with [neratinib (0.5 μM) + valproate (250 μM)]. After an additional 24h cells were treated with live/dead reagent and the percentage cell death under each condition determined. (n = 3 +/-SEM) * p < 0.05 less killing compared to siSCR cells.