Inhibition of cysteine cathepsins during EMT impaired TGFβ-1 induced invasiveness of malignant cells. (A) Experimental setup: Cells were either untreated or treated with TGFβ-1 for four days in presence of E64d or solvent control (“ctrl”) for three days. At day three E64d was removed. At day 4 cells were trypsinized and directed migration or invasion through Cultrex®-coated membranes were analyzed for 24 h by RTCA real time trans-well assays in absence of E64d. (B) Cysteine cathepsin activity measured by z-Phe-Arg-AMC hydrolysis in NMuMG and iPL32 whole cell lysates after three days E64d treatment and one day E64d withdrawal. (C-F) Migration and Invasion of NMuMG (C,D) and iPL32 cells (E,F). Cell indexes as function of time for the 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).