Fig. 1. Decreased cell aggregation and sensitivity to EGTA-induced detachment in primary Timp3^–/– MEFs. (A) Representative examples of aggregation assay with independent MEF preparations. WT aggregates (arrows) were evident at 15 minutes and large aggregates seen at 45 minutes. Timp3^–/– MEF aggregates (arrows) did not form until 45 minutes and were much smaller in size (fewer cells) than WT aggregates. Bars, 70 µm. (B) Representative images of fibroblast monolayers in a Ca²⁺ chelation assay. Timp3^–/– MEFs detached before WT controls, as assessed by Hoechst staining and fluorescence microscopy of the cells remaining on the coverslips after the indicated time of EGTA treatment. Bars, 70 µm. (C) The number of viable cells in the supernatant following EGTA-induced detachment, assessed using Trypan Blue exclusion, showed higher numbers of Timp3^–/– cells at all incubation times (^*P<0.04; , WT; , Timp3^–/–). Student's t-test was performed to assess statistical significance between WT and Timp3^–/–. Values are expressed as mean ± s.e.m. (D) Western blot analyses of whole cell lysates from three of the five independent clones of WT and Timp3^–/– MEFs, probed with the indicated antibodies. Amido Black staining was used to assess protein loading.