Mks use podosomes to cross a native basement membrane. Mks were spread for 3 hours on a native basement membrane fixed and stained for F-actin (red) and collagen IV (green). The right column of pictures shows the merge of both channels. Representative images are shown for (A) 0.1% DMSO used as control and (B) 20 μM CK666. Pictures show slice of taken z-stack from each treatment and the corresponding cross section, XYZ perspective. (C) The percentage of actin protrusions crossing the basement membrane was analyzed for different inhibitors; 20 μM CK666, 5 μM GM6001, 10 μM blebbistatin, 5 μM GM6001/10 μM blebbistatin, and WASp^−/− Mks. Yellow arrowhead indicates actin-rich protrusion crossing the membrane. Red line represents position of cross section. The cross sections of at least 10 different cells were analyzed per experiment. Data are representative of at least 3 independent experiments. Images were taken with a confocal microscope using a 40× objective. Scale bars represent 20 μm. Statistical analysis was done by performing a 1-way analysis of variance. Asterisk indicates significant difference with a P value < .05. Error bars indicate ±SEM.