Inhibition of cysteine cathepsins during migration/invasion reduced invasion of TGFβ-1 transformed malignant cells. (A) Experimental setup: Cells were treated without or with TGFβ-1 for four days in the absence of E64d. At day four cells were incubated with E64d or solvent control (“ctrl.”) for one hour. Thereafter cells were trypsinized and migration and invasion were analyzed for 24 h by RTCA real time trans-well assays in presence of E64d. (B) Cysteine cathepsin activity measured as z-Phe-Arg-AMC hydrolysis in NMuMG and iPL32 whole cell lysates after 1 h E64d treatment. (C-F) Migration and invasion of (C,D) NMuMG and (E,F) iPL32 cells. Graphs show the cell indexes of triplicates of representative experiments (left panels); statistical analysis of independent experiments (right panels) calculated as the slope of cell index between the time points marked in the time-curves, normalized to “+TGFβ-1 ctrl.”, shown as the mean ± SEM (n = 3 for NMuMG and n = 5 for iPL32 cells, *p ≤ 0.05, **p ≤ 0.01).