Fig. 4. Effects of forced expression of RECK or MMP on chondrogenic differentiation of ATDC5 cells. (A) The cells transfected with the control vector (left panels) or the vector expressing human RECK (right panels) were incubated for the indicated times and then stained with Alcian Blue. Bars, 2 mm. (B) Expression of chondrogenic differentiation markers in the transfectants. Total RNA (20 µg) extracted from the transfectants at the indicated times (bottom labels) was analyzed by RNA blot hybridization using the indicated probes (top labels). (C) Effects of forced RECK expression on the level of secreted gelatinases. Conditioned media (7 hours conditioned) prepared from vector-(V) or RECK-transfected ATDC5 cells (R) at the indicated times were analyzed by gelatin zymography. The positions of the pro-MMP9, pro-MMP2, and intermediate-MMP2 bands are indicated. The amounts of samples applied were adjusted between V and R so as to represent equal cell number. Day 7 samples were five times more concentrated than day 3 samples on a per-cell basis. (D) Effects of forced expression of MMPs on cellular condensation of ATDC5 cells. ATDC5 cells were stably transfected with a mammalian expression vector (pcDNA3.1/Hygro(+); Invitrogen) or the vector containing mouse Mmp2, mouse Mmp9, or human MT1-MMP cDNA. Phase-contrast micrographs of vector- or MMP-transfected ATDC5 cells incubated for 5 days are shown. Expression of MMP in each transfectant was confirmed by northern blot hybridization and gelatin zymography. Note the accelerated cellular condensation in Mmp9-transfected cells and MT1-MMP-transfected cells. Bars, 100 µm.