Fig. 2. Expression patterns of MMP, collagens and RECK during cartilaginous nodule formation by ATDC5 cells. (A) Temporal expression patterns of Mmps and Reck in differentiating ATDC5 cells. Total RNA (20 µg) extracted from ATDC5 cells that had been incubated for the indicated times was analyzed by RNA blot hybridization using indicated probes. (B) Collagenase activity expressed by differentiating ATDC5 cells. ATDC5 cells were incubated for the indicated times and then overlaid with semi-solid medium containing reconstituted type I collagen and DQ collagen. After an additional 24 hours incubation, morphology (DIC) and collagenolytic activity (green DQ-fluorescence) were recorded with a confocal microscope. Bars, 100 µm in the day-5 and day-10 panels; 200 µm in the day-24 panel. Arrowheads indicate the rim of a large nodule. (C) Sensitivity of the DQ-fluorescent signals to an MMP inhibitor GM6001. The experimental conditions were the same as in B, day 24 except that the cells were exposed to GM6001 (100 µM; bottom panels) or an inactive analogue (100 µM; control) for the last 36 hours (i.e. 12 hours before overlay plus 24 hours after overlay). Bars, 100 µm. (D) Temporal expression pattern of type I collagen mRNA in differentiating ATDC5 cells. The same blot used in A was re-probed with a type I collagen cDNA. (E and F) Localization of type I and type II collagen at cartilaginous nodules formed by ATDC5 cells. ATDC5 cells incubated for 15 days were fixed and stained with anti-RECK (red) plus anti-type I collagen (green; E) or anti-RECK (red) plus anti-type II collagen (green; F). Reconstituted z-axis images along two cutting lines (a,b) are shown in the top and right panels, respectively. Bars, 100 µm in E,F.