Figure 8.
Figure 8. Impaired cell–cell communications via gap junction associated with altered Cx43 expression in type XII collagen–null osteoblasts. (A–C) Analysis of Cx43 (Cx43; A), Cad2 (Cad2; B), and Cad11 (Cad11; C) mRNA expression in cultures of primary osteoblasts by quantitative real-time PCR normalized to Gapdh expression. Each group represents the mean of triplicate determinations. (A) Cx43 expression is significantly decreased at every time point in Col12a1−/− osteoblast cultures. (B) No significant difference between genotypes is detected in Cad2 expression. (C) Cad11 expression is decreased in early stages in Col12a1−/− osteoblast cultures but not in late stages. **, P < 0.01. (D) Immunoblot analysis of Cx43 in cultures of primary osteoblasts. β-Actin is used as an internal control. (E) The relative band density of Cx43 normalized to β-actin. Each group represents the mean of triplicate determinations. Cx43 expression is decreased in Col12a1−/− osteoblasts. (F) Immunoblot analysis of Cx43, Cad2, and Cad11 in P30 femurs from Col12a1+/+ (n = 2) and Col12a1−/− (n = 3) mice. Cx43 and Cad11 expression is decreased in Col12a1−/− femurs. Molecular mass is indicated in kilodaltons. (G) Dye coupling study in primary osteoblasts. Calcein AM– and Cell Tracker–labeled osteoblasts (DCC, dye-carrying cells) were seeded onto confluent osteoblasts (host cells) and analyzed by confocal microscopy. The dashed lines indicate the outline of host cells. High magnification of the site of dye-carrying wild-type osteoblasts (+/+) attaching to the wild-type host osteoblasts demonstrates transfer, i.e., the green dots (arrows) to the host cell. Although the Col12a1−/− osteoblast is attaching to the Col12a1−/− host cell, calcein dye stays within the donor cell (i.e., no transfer is observed). Bars: (top) 25 µm; (bottom) 10 µm. (H) The number of dye-transferred cells per total parachuted cells was determined in 10 random images acquired from each well (24-well plate), and 6 wells were used per experimental group. As compared with dye-carrying wild-type cells (+/+) on wild-type host cells, the percentage of dye-transferred cells is significantly decreased when either dye-carrying or host cells are Col12a1−/−. A reduction of ∼40% is detected when compared with wild-type versus wild-type and Col12a1−/− versus Col12a1−/− cells. *, P < 0.05; **, P < 0.01. Error bars are mean ± SD.

Impaired cell–cell communications via gap junction associated with altered Cx43 expression in type XII collagen–null osteoblasts. (A–C) Analysis of Cx43 (Cx43; A), Cad2 (Cad2; B), and Cad11 (Cad11; C) mRNA expression in cultures of primary osteoblasts by quantitative real-time PCR normalized to Gapdh expression. Each group represents the mean of triplicate determinations. (A) Cx43 expression is significantly decreased at every time point in Col12a1−/− osteoblast cultures. (B) No significant difference between genotypes is detected in Cad2 expression. (C) Cad11 expression is decreased in early stages in Col12a1−/− osteoblast cultures but not in late stages. **, P < 0.01. (D) Immunoblot analysis of Cx43 in cultures of primary osteoblasts. β-Actin is used as an internal control. (E) The relative band density of Cx43 normalized to β-actin. Each group represents the mean of triplicate determinations. Cx43 expression is decreased in Col12a1−/− osteoblasts. (F) Immunoblot analysis of Cx43, Cad2, and Cad11 in P30 femurs from Col12a1+/+ (n = 2) and Col12a1−/− (n = 3) mice. Cx43 and Cad11 expression is decreased in Col12a1−/− femurs. Molecular mass is indicated in kilodaltons. (G) Dye coupling study in primary osteoblasts. Calcein AM– and Cell Tracker–labeled osteoblasts (DCC, dye-carrying cells) were seeded onto confluent osteoblasts (host cells) and analyzed by confocal microscopy. The dashed lines indicate the outline of host cells. High magnification of the site of dye-carrying wild-type osteoblasts (+/+) attaching to the wild-type host osteoblasts demonstrates transfer, i.e., the green dots (arrows) to the host cell. Although the Col12a1−/− osteoblast is attaching to the Col12a1−/− host cell, calcein dye stays within the donor cell (i.e., no transfer is observed). Bars: (top) 25 µm; (bottom) 10 µm. (H) The number of dye-transferred cells per total parachuted cells was determined in 10 random images acquired from each well (24-well plate), and 6 wells were used per experimental group. As compared with dye-carrying wild-type cells (+/+) on wild-type host cells, the percentage of dye-transferred cells is significantly decreased when either dye-carrying or host cells are Col12a1−/−. A reduction of ∼40% is detected when compared with wild-type versus wild-type and Col12a1−/− versus Col12a1−/− cells. *, P < 0.05; **, P < 0.01. Error bars are mean ± SD.

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