Fig. 4. rpS6 phosphorylation is neither necessary nor sufficient for the growth inhibitory effects of the combined inhibition of Src and the MAPK pathway.

a Western blot analysis was performed on whole cell lysates from the 8505C or C643 cell line expressing a doxycycline inducible P70S6K T389 ΔCT construct. Cells were treated with or without doxycycline for 24 h prior to treatment. After 24 h the cells were treated with either DMSO, 100 nM trametinib, 100 nM dasatinib, or the combination of dasatinib and trametinib. Lysates were analyzed for the indicated antibodies. Quantification was performed using the Odyssey Image Studio V.4.0 Software. Data as means±SEM (n = 3). b Clonogenic growth was detected by crystal violet staining in the 8505C or C643 cell lines expressing the doxycycline inducible P70S6K T389 ΔCT construct. Cell lines were treated with or without doxycycline upon plating. 24 h later the cell lines were treated with either DMSO, 100 nM trametinib, 100 nM dasatinib, or the combination for 6 days. Following 6 days of treatment, the cells were released for an additional 7 days. c Western blot analysis was performed on whole cell lysates from the parental 8505C or parental C643 cell lines treated with either DMSO, 100 nM trametinib, 100 nM dasatinib, or 1 μM Everolimus for 24 h for the indicated antibodies. Quantification was performed using the Odyssey Image Studio V.4.0 Software. Data as means±SEM (n = 3). d Clonogenic growth was detected by crystal violet staining in the 8505C and C643 cell lines. Cell lines were treated for 24 h with either DMSO, 100 nM trametinib, 100 nM dasatinib, dasatinib+trametinib, or 1 µM everolimus. Colony area signal intensity was measured using Odyssey CLx imager (Li-Cor), and presented as percent fold change relative to the DMSO treated wells. Data as means±SEM (n = 3)