EGF activates Rac1 in single cells and not collectively invading cells. (A) Flow cytometric surface protein quantification of integrin α6 (Itga6) in leaders and singles. Dotted line denotes the unstained sample for each subpopulation. (B) Time course measurement of relative Rac1 activity in leaders and singles upon 50 ng/ml hEGF treatment (n = 3, ***P < 0.001, ****P < 0.0001). Mean ± SEM is shown. (C) Volcano plot of differentially secreted peptides extracted via LC-MS/MS from CM from leaders with Lama3 WT and KO (n = 3). Red circles denote peptides differentially secreted in Lama3 WT leaders and green circles denote peptides differentially secreted in Lama3 KO leaders (|Student’s T test Difference| >1, P value <0.05).