Characterization of MDA-MB-231 and MDA-MB-231-BM cells with regard to hyaluronan production; HAS, HYAL, and CD44 mRNA expression; and invasive behavior. A, hyaluronan production. Cells (2 × 105) were seeded in a 6-cm dish and grown for the indicated time periods. Conditioned media were collected, and the hyaluronan amount was determined. The graph shows the average ± S.D. of triplicates. B, assembly of pericellular matrices. MDA-MB-231 and MDA-MB-231-BM cells (2 × 104 cells/well) were seeded in 6-well plates, and their hyaluronan coat was determined by a particle exclusion assay after 24 h. Photographs were taken with a Zeiss Axiovert phase-contrast microscope. Scale bars = 25 μm. C, HAS, HYAL, and CD44 mRNA expression. Cells (2 × 105 cells/well) were grown in 12-well plates for 24 h. The expression levels relative to GAPDH of HAS1, HAS2, HAS3, HYAL1, HYAL2, CD44s, CD44v3, and CD44v6 mRNAs were determined by real-time PCR as described under “Materials and Methods.” Results are means ± S.D. of triplicate determination. *, p < 0.05 (Student's t test, significant difference compared with MDA-MB-231 cells). D, in vivo-like invasion assay. Cells (1.5 × 105 cells/well, 48-well plates) were embedded in 100 μl of growth factor-reduced Matrigel/dish and incubated for 6 or 24 h. Representative pictures were taken with a Zeiss Axiovert phase-contrast microscope. Scale bar = 100 μm. E, expression of MMP2, MMP7, MMP9, and MT1-MMP proteins. Cell lysates and conditioned media were subjected to SDS-PAGE using 10% polyacrylamide gels, followed by immunoblotting with antisera against MMP2, MMP7, MMP9, MT1-MMP, and actin as described under “Materials and Methods.” The data presented are from a representative experiment of two performed with similar results.