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. 2018 Jan 2;118(3):366–377. doi: 10.1038/bjc.2017.421

Figure 4.

Figure 4

Adaptive rewiring upon Mtor deletion in murine PDAC cells. (A) PPT4-ZH363-MtorΔE3/lox or control PPT-c1674 cells were treated with vehicle or 4-OHT (600 nM) over 8 days. Western blot determines phosphorylation and expression of AKT (Thr308 and Ser473) as well as ERK (Thr202/Tyr204). Same lysates were blotted to different membranes and controlled by pan-AKT or pan-ERK for equal loading. (B) PPT4-ZH363-MtorΔE3/lox or control PPT-c1674 cells were treated with vehicle or 4-OHT (600 nM) over 8 days. Quantification of independent biological replicates of phosphorylation of AKT at Thr308 and Ser473 as well as ERK at Thr202 and Tyr 204. The ratio of the depicted phospho-protein to the pan-protein in untreated control PPT-c1674 cells was arbitrary set to one. The number of replicates is depicted. *P-value of a paired Student’s t-test<0.05. (C) Indicated cells were treated as in (A). Western blot determines phosphorylation of Mek1/2 at Ser217/221 and expression of Mek1/2. Actin: loading control. Experiment was performed once in PPT-c1674 cells and twice (#1, #2) in PPT4-ZH363-MtorΔE3/lox cells. All replicates were blotted to the same membrane. The ratio of phosphorylated to pan-Mek1/2 was determined and arbitrary set to 1 in vehicle-treated cells. The relative Mek1/2 phosphorylation is depicted. (D) Indicated cells were treated as described in (A). Afterwards, cells were treated for 6 h with INK-128 (2 μM), PD-325901 (2 μM), GDC-0941 (4 μM) or MK-2206 (4 μM). Western blot of phospho- (Ser473) and pan-AKT, phospho-S6 (Ser235/Ser236), phospho- (Thr202/Tyr204) and pan-ERK (n=2). (E) PPT4-ZH363-MtorΔE3/lox cells were treated over time as indicated with INK-128 (0.5 μM). Western blot of phospho- (Thr308 and Ser473), pan-AKT, phospho-S6 (Ser235/Ser236), phospho- (Thr202/Tyr204) and pan-ERK. Heat-shock protein 90 (HSP90) and actin: loading control. Same lysates were blotted to different membranes (n=2). (F) PPT4-ZH363-MtorΔE3/lox cells were treated over time as indicated with AZD2014 (0.5 μM). Western blot of phospho-Ser473 and pan-AKT, phospho-S6 (Ser235/Ser236), HSP90: loading control. Same lysates were blotted to different membranes (n=3). For (E) and (F) medium with fresh inhibitors or vehicle was replaced each day.