Figure 2

TG2 inhibits the invasion of MDA-MD-231 cells through a Matrigel-coated Transwell membrane. A, Matrigel contents were incubated with buffer alone (control) or buffer containing the purified guinea pig liver TG2 protein (6 μg/0.75 ml) in the presence (TG2 + Ca²⁺) or absence (TG2 alone) of 5 mM Ca²⁺ at 37°C for 15 minutes before being coated onto the Transwell membranes. The MDA-MB-231 cells were compared for their ability to invade through the TG2-pretreated or untreated Matrigel-Transwell membranes. Representative fields with cells that migrated under the membrane were photographed. B, Matrigels (0.75 ml) containing increasing amounts of the purified TG2 protein were preincubated (37°C, 15 minutes) with 5 mM Ca²⁺. Another tube containing Matrigel plus 6 μg of TG2 protein was incubated in parallel, but without Ca²⁺, to serve as control. Matrigel contents pretreated with various amounts of TG2 were layered over the Transwell membranes and compared for their ability to support the invasion of MDA-MB-231 cells. Ten fields were counted randomly under the microscope for the number of cells that had migrated through the Matrigel and were plotted as an average number of cells ± SD per field. C, Equal volumes of Matrigel (0.75 ml) were incubated under conditions described in legend B with buffer alone or buffer containing varying concentrations of purified TG2 in the presence (+) or absence (-) of 5 mM Ca²⁺. Forty μl reactants, each were fractionated on SDS-PAGE. The gel was stained and viewed for TG2-induced changes in protein bands after destaining in methanol/acetic acid solution. The thin arrow indicates a prominent band that disappears in the presence of an enzymatically active TG2; whereas thick arrow indicates the appearance of a prominent band in the presence of active TG2. D, Basal levels of MMP-1 and MMP-2 in MDA-MB-231 cells as determined by zymogram performed on the supernatants collected from cultured cells (80% confluent in 6-well plate, incubated in 1 ml serum-free medium overnight) or by RT-PCR, using MMP-1 specific primers, as described in Materials and Methods.