Fig. 5. Effects of Reck gene knockdown on cartilaginous nodule formation by ATDC5 cells. (A) ATDC5 cells were stably transfected with the control vector (V) or the vector expressing shRNA against Reck (S). Expression of RECK in these cells were analyzed by immunoblot assay (top panel) and RNA blot hybridization (middle panel). Total proteins (30 µg) harvested on day 7 and total RNA (20 µg) harvested on day 4 were used. The number under each lane indicates relative band intensity. (B) Phase-contrast micrographs (top 2 rows) and Alcian blue-stained foci (third row) of the vector-transfected cells (left panels) or RECK-siRNA-transfected cells (right panels) incubated for the indicated times. Bars, 100 µm (Day 12); 250 µm (Day 15) and 400 µm (Day 24). (C) The number and morphology of foci. After incubation for the indicated times, the number of all visible foci (Total foci) and the number of highly refractile foci (e.g. in B, Vector Day 15, arrow), which represent ECM-rich cartilaginous nodules (Shukunami et al., 1998), were scored under a microscope. Values are mean ± s.e.m. from quadruple dishes. (D) Immunofluorescent staining of foci at day 15. The cells were doubly stained with anti-type II collagen (green) and anti-RECK (red) antibodies. Morphology (DIC), type II collagen signals (Col-II), and RECK signals (RECK) around typical foci were recorded under the same microscopic conditions. Bars, 100 µm. The findings were consistent between two separate experiments using independently derived transfectants.