Fig. 3. Effects of forced RECK expression on nodule formation by ATDC5 cells. (A) ATDC5 cells were stably transfected with a mammalian expression vector pCXN2 (V) (Niwa et al., 1991) or the vector expressing human RECK (R). Expression of RECK protein (top panel) and RECK mRNA (middle panel) in the pooled transfectants harvested under the proliferative conditions was analyzed by immunoblot assay (30 µg protein per lane; top panel) and RNA blot hybridization (10 µg total RNA per lane; middle and bottom panels), respectively. Relative band intensity is shown under each lane. (B) Growth curves of the transfectants. The plating density at day 0 was 3x10⁴ cells per 35-mm dish. The data are average values from duplicate dishes. (C) Phase-contrast micrographs of the vector-transfected cells (left panels) and RECK-transfected cells (right panels) incubated for the indicated times. Bars, 300 µm. Arrows indicate compact nodules. (D) Number of foci (areas of cell condensation) formed by the vector-transfected cells (hatched bars) and RECK-transfected cells (white bars) after incubation for the indicated times. The data are given as mean ± s.e.m. of quadrate dishes. Consistent results were obtained in two separate experiments using the same set of transfectants and in another experiment using retroviral vectors for gene transfer.