Figure 3.

Analysis of in vitro attributes of tumor progression. (a) Equal amounts (20 μg) of protein extracts from mammalian relative of DnaJ (MRJ) long isoform (MRJ [L]) expressors (M) of MDA-MB-231 and MDA-MB-435 were compared with the parent (P) and vector control (V) for the level of MRJ(L). MRJ(L) migrates at the apparent molecular weight of 38 kDa. Equal loading was confirmed by comparable β-actin signal. (b) MDA-MB-231-MRJ(L), referred to as 231-MRJ(L), and MDA-MB-435-MRJ(L), referred to as 435-MRJ(L), exhibited decreased migration compared with the corresponding vector controls (231-vec and 435-vec) in wound healing assay. Cells were cultured to confluence on premarked six-well plates. A central linear wound was made with a 200 μl sterile pipet tip. Phase micrographs of the wound cultures were taken at 0 and 16 hours. The photographs were analyzed by measuring the distance from the wound edge of the cell sheet to the original wound site. The dotted white lines in the photomicrographs indicate the original position of the wound. Migration activity was calculated as the mean of the distance between the edges in 12 independent fields per well. Each test group was assayed in triplicate, and the results are expressed relative to vector control cell migration. *P < 0.05. (c) MDA-MB-435-MRJ(L) exhibited reduced anchorage-independent growth compared with vector control. Cells (2 × 10³) suspended in 0.35% agar were plated onto a layer of 0.75% bactoagar in Dulbecco's modified Eagle's medium/F12 (5% fetal bovine serum) in six-well tissue culture dishes. Visible colonies (> 50 cells) were counted after 15 days with the aid of a dissecting microscope. The results are expressed as mean number of colonies ± standard error of the mean. *P < 0.05. (d,e) MRJ(L) expressors of MDA-MB-231 and MDA-MB-435 exhibit significantly reduced ability to migrate through transwell (panel d) and are retarded in terms of their ability to invade through Matrigel™- coated filters (panel e). Migration and invasion assays were conducted using 8 μm polyethylene terphthalate filters, as previously described [18]. Cells migrated to the lower sides of the transwell were stained using Diff-Quik^® reagent and the cells were counted under a microscope. Each test group was assayed in triplicate. Four different fields of each insert were photographed; each field was divided into quadrants and cells in diagonally opposite quadrants were counted.