Fig. 3
Functional annotation of Src-regulated kinases. a Monolayer (2D) proliferation of MCF-10A control (_Ctrl) and MCF-10A_Src cells, −/+EGF. MTS assays were undertaken in 96-well plate as described in Methods. Error bars represent s.d. from five replicates. b Growth characteristics of MCF-10A_Ctrl and MCF-10A_Src cells in 3D Matrigel culture, −/+EGF. Cells were plated in 3D culture −/+EGF in a 96-well plate as described in Methods. Acini were imaged (Scale bar represents 50 μm) for both +EGF and −EGF conditions (top panels) and for the latter condition, the size of >100 acini were measured and plotted on a box and whisker graph, with the center line indicating the median, the upper and lower bounds of box indicating the 25th and 75th percentiles of the data, and the upper and lower whiskers indicating the 5th and 95th percentiles of the data (bottom panel). ****p < 0.0001 by Mann–Whitney test. c Kinase-dependency of control and Src-transformed cells under –EGF conditions. siRNA SMARTpools of targets were robotically reverse transfected in duplicate 96-well plates at 40 nM and cell viability measured 96 h post-transfection. Relative viability values for MCF-10A_Ctrl (Ctrl) and MCF-10A_Src (Src) cells were plotted. Each dot indicates a kinase screened in both Ctrl and Src cells. Red line is the line of equality (y = x). Relative viability <40% in both Ctrl and Src cell lines is defined as “essential for both”. Relative viability <40% in Ctrl cells, and >60% in Src cells is defined as “essential for Ctrl, dispensable for Src”. No kinases were essential for Src cells, dispensable for Ctrl. d Kinases required preferentially by controls. Targets identified in (c) as “essential for Ctrl, dispensable for Src” were plotted. Error bars represent s.e.m. from two biological replicates. e Dependency on MAP4K5 is growth factor- and cell type-dependent. Viability is expressed relative to non-targeting control (NTP) which was arbitrarily set at 100%. Error bars represent s.e.m. from three or four biological replicates. *p < 0.05, ****p < 0.0001 by Student’s t-test
