Fig. 4. Delayed differentiation of Cx43-deficient osteoblasts. (A) X-gal staining of post-confluent ColCre;Cx43^–/fl calvaria cells grown in mineralizing medium, showing blue staining becoming stronger with time in culture. (B) Western analysis demonstrated barely detectable Cx43-specific bands in lysates of conditionally deleted cells, and reduced abundance of Cx43 in heterozygous equivalent cells. (C) Abundance of Cx43 mRNA, assessed by real-time PCR, was reduced in ColCre;Cx43^–/fl calvarial cells relative to wild-type equivalent cells after 3 weeks in culture (n=3). (D) Development of alkaline phosphatase activity was significantly reduced in calvarial cells derived from ColCre;Cx43^–/fl mice 2 weeks post-confluence (n=4). (E) The abundance of mRNA transcripts for other osteoblast-specific genes was reduced by more than 50%, as measured by real-time PCR, relative to wild-type equivalent cells (*P<0.05 versus Cx43^+/fl; one-way ANOVA; n=3). (F) Representative results of von Kossa stain of post-confluent calvaria cells and (G) quantitative data on three different preparations showing delayed in vitro matrix mineralization by ColCre;Cx43^–/fl calvaria cell cultures (*P<0.05 versus Cx43^+/fl; Tukey test; n=3).