Figure 1. Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent.

(A) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. (B) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. (C) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. (D) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.(E) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). (F) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. (G) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n>30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.