Fig 2: Synergy of RSK and SRC pathway activating RTKs induces ATP in consequence of low-dose BMS777607 treatment in Caliper and MDA-MB231. (A + B) Cells were cultivated in 3D in the presence of increasing BMS777607 concentrations in a range between 0.001 and 12.5 µm. ATP measurements were taken after 3 days. DMSO control values were set to 100% and indicated by a doted horizontal line. Nanomolar concentrations of BMS777607 increase significantly the ATP content of Caliper cells and to a lower extent of MDA-MB231 cells, in contrast to micromolar concentrations. Percentages of ATP inhibition were calculated from at least three independent experiments. (C) MDA-MB231 and Caliper cells were treated with 0.39 µm BMS777607 for 3 days. The western blot analyses display the induction of RSK S380 phosphorylation in both cell lines compared to untreated cells. MDA-MB231 cells activated all three TAM members, autocrine GAS6 and PROS1, whereas Caliper recruited AXL, MERTK, and GASA6 in consequence of BMS777607 treatment. (D) Further western blot analyses show an enhancement of SRC and SRC-dependent MET Y1349 and AXL Y779 phosphorylation with a slightly enhanced AKT S473 signal in both cell lines. A significant ERK5 phosphorylation is only detectable in Caliper cells in accordance with a stronger pMET Y1349 and pSRC Y416 activation compared to MDA-MB231 cells. (E and F) Caliper cells were cultivated in 3D in the presence of 0.001–12.5 µm of 84 111 and sunitinib. ATP measurements were taken after 3 days. DMSO control values were set to 100% and indicated by a doted horizontal line. A significant ATP increase was induced by nm concentrations of sunitinib in contrast to nm concentrations of 84 111. An inhibition of ATP by sunitinib was detectable at concentrations = 1.56 µm and at concentrations = 0.097 µm by 84 111. (G) Caliper cells were treated for 3 days either with 0.39 µm 84 111 or with sunitinib. Western blot analyses show the RSK and SRC-dependent pathway activation by sunitinib compared to untreated cells. 84 111 reduced AKT-mTOR phosphorylation due to inhibition of SRC as well as MET Y1349 and AXL Y779 phosphorylation, but had no effect on RSK phosphorylation.