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. Author manuscript; available in PMC: 2021 Feb 9.
Published in final edited form as: Matrix Biol. 2020 Oct 20;95:52–67. doi: 10.1016/j.matbio.2020.10.004

Fig 6. Collagen XII regulates establishment of a communicating tenocyte network.

Fig 6.

(A) Connexin 43 immunolocalization was performed in primary tenocytes obtained from FDLs of Col12a1+/+ and Col12a1−/− neonatal mice. Tenocyte structure was visualized after staining of the cytoplasmic filamentous actin network with Alexa 594 Phalloidin (red) and nuclei were stained with DAPI (blue). Col12a1+/+ tenocytes are dendritic and connexin 43 (green) is localized where tenocyte processes interacted with other processes from adjacent tenocytes. In contrast, Col12a1− /− tenocytes have altered shapes with no clear processes, and no specific localization of connexin 43 is observed. Scale bars 25 μm. (B) Connexin 43 and cell adhesion molecules were analyzed by Western blotting in P10 and P30 FDLs from Col12a1+/+ and Col12a1−/− mice. Connexin 43 and cadherin 11 expression is equivalent between Col12a1+/+ and Col12a1−/− at both P10 and P30. N-cadherin expression is similar between genotypes at P4, but slightly decreased in P30 Col12a1−/− FDLs when compared to Col12a1+/+. b-actin was used as an internal control. (C,D) Collagen XII localization was analyzed in primary tenocytes obtained from Col12a1+/+ FDLs. (C) Immunostaining for collagen XII (green), phalloidin (red) and DAPI (blue) was analyzed in 30%, 70% and 100% confluent cell cultures. At 30% confluence, few intercellular connections are formed. Collagen XII is detected as punctate structures on the tenocyte surface (arrowheads). At 70% confluence, intercellular connections begin to form. Collagen XII is localized along the processes connecting neighboring cells (double arrows). At 100% confluence, collagen XII forms a network connecting neighboring cells. (D) Collagen XII forms bridges between neighboring cells before they establish physical connection. Scale bars 100 μm.