Fig. 1. Abundant expression of Reck in cartilage. (A) Mouse embryo multiple tissue northern blot (Clontech) was hybridized sequentially with cDNA probes for Reck and Gapdh (loading control). Each lane contained 2 µg of poly(A)⁺ RNA from embryos at the indicated stage (days after gestation). (B-G) Detection of Reck mRNA in mouse embryos by in situ hybridization. Sagittal sections of an E13.5 embryo were hybridized with Reck antisense (B) or sense (C) riboprobe. Magnified views of the boxed areas in B are shown in D and E (^*), respectively. The areas containing ribs (F) and a femur (G) in a section of E16.5 embryo hybridized with Reck anti-sense probe are also shown. Northern blot data (not shown) indicated that the apparently strong signals in the liver (labeled `L' in panel B and F) are non-specific. Bars, 1.28 mm in B and C; 200 µm in D and E; 320 µm in F and G. (H-J) Detection of Reck mRNA in cultured cells by RNA blot hybridization. Total RNA extracted from the following cultured cells was analyzed by RNA blot hybridization using the Reck cDNA probe. (H) Lane 1: H4-1 (a primary culture derived from human bone marrow containing the cells capable of differentiating into chondrocytes) (Imabayashi et al., 2003; Mori et al., 2005). Lane 2: DEC (chondrocytes obtained from human cartilage) (Imabayashi et al., 2003). Lane 3: the human fibrosarcoma cell line HT1080-transfected with control vector (negative control). Lane 4: HT1080 transfected with RECK expression vector (positive control). (I) Lanes 1 and 2: longer exposure of lane 1 and 2 in H, (under the same conditions as lanes 36). Lane 3: KUM9 (multipotential progenitor cells derived from mouse bone marrow and capable of differentiating into osteocytes, adipocytes, myocytes and neurons) (Kohyama et al., 2001). Lane 4: KUSA-A1 (osteoblasts derived from mouse bone marrow) (Kohyama et al., 2001). Lanes 5 and 6: MDBM (osteoclast progenitor cells derived from mouse bone marrow) and RANKL-treated MDBM (mature osteoclasts), respectively (Takeshita et al., 2000). The amount of total RNA loaded was 10 µg for lanes 1-4 and 4 µg for lanes 5-6. Patterns of ribosomal RNA bands are also shown (bottom panels). (J) Relative intensity of the bands on the blot shown in I. The band intensity determined by densitometry and normalized against the RNA amount is presented as a bar graph for direct comparison. (K) Temporal expression pattern of Reck mRNA in ATDC5 cells during chondrogenic differentiation in vitro. Total RNA (20 µg) from ATDC5 cells that had been allowed to differentiate in culture for the indicated times (days) was subjected to RNA blot hybridization using a Reck cDNA probe (top panel). The same blot was re-probed with a type II collagen cDNA (middle panel; Col-II) to monitor chondrogenic differentiation.