Figure 5.

Nonadherent tuberin-null cells are invasive. (A) Collagen gel invasion assays show that N-Adh Tsc2^−/− cells are invasive (right panel). The white outline depicts the boundary of the collagen gel area cleared by N-Adh Tsc2^−/− invasive cells. The left panel depicts an Adh Tsc2^−/− cell attached to the collagen matrix (denoted by black arrowhead) without any effects on the underlying collagen gel. The middle panel shows the same cell enlarged 3×. (B) Feeder cell invasion assays demonstrate that N-Adh Tsc2^−/− cells invade epithelial cell layers and secrete cleaved, active MMP7. Lane 3 shows the expression of active MMP7 from immunoprecipitated media at 2 days postaddition. Lane 1, feeder cells; lane 2, feeder cells + Adh Tsc2^−/− cells; lane 3, feeder cells + N-Adh Tsc2^−/− cells; lane 4, feeder cells + Tsc2^+/+ cells. Upper confocal images depict a N-Adh Tsc2^−/− cell colony invading epithelial cell layers by Day 2 (2 days). Lower images show the continued invasion of N-Adh Tsc2^−/− cells through a feeder layer by Day 5 (5 days). Green, smooth muscle actin staining of the N-Adh Tsc2^−/− cells; red, nuclei visualized with TOPRO3 in all cells. (C) Inhibition of MMP7 activity blocks the invasion of N-Adh Tsc2^−/− cells in collagen gel invasion assays. N-Adh Tsc2^−/− cells were incubated with a combination of two MMP7 antibodies, each at the following concentrations: 0, 0.25, 1.0 μg/ml. The results shown represent the average number of collagen areas cleared by invasive colonies counted per field (with a minimum of five fields counted) of two independent experiments. *P < 0.05. (D) Analysis of lymphangioleiomyomatosis tissue samples demonstrates the presence of cleaved β-catenin and MMP7 (lanes 3–9). Immunoblot analyses of lung lysates using the indicated antibodies. Normal lung tissues are represented in lanes 1 and 2.