Fig. 5

The effects of DKK1 secreted by fibroblasts on the osteoblast differentiation of MSCs. A Heatmap with mRNA-based expression of representative genes (cytokines) in fibroblasts either cultured alone or co-cultured with MSCs. The gene names were shown on the right of the heatmap. B Histogram showed mRNA-based expression of representative genes (cytokines) in fibroblasts either cultured alone or co-cultured with MSCs. C Conditional media obtained from either 1 × 10⁴ fibroblasts or MSCs were collected and analysed with Elisa assay to determine the concentrations of DKK1 (*P < 0.05). D After 1 × 10⁴ MSCs were treated with conditional media from fibroblasts, 10 ng/mL DKK1 or 5 µM gallocyanine (DKKi) for 7 days, cells were harvested and subjected to ALP staining. After the cells were treated with conditional media from fibroblasts, 10 ng/mL DKK1 or 5 µM gallocyanine for 14 days, cells were harvested and subjected to Alizarin red staining. 200 ng/mL BMP2 was utilized to induce osteoblast differentiation of MSCs. The representative images of the wells and cells were presented. Scale bars, 100 μm. E, F The percentages of ALP⁺ cells (E) and Alizarin red⁺ areas (F) in D were quantitatively analysed. The average percentages of ALP⁺ MSCs and Alizarin red⁺ areas were calculated from at least three fields and presented (^#represented comparison between indicated group and negative control; ^#P < 0.05; ^##P < 0.01; **P < 0.01). G MSCs were treated as indicated in D. Subsequently, the cells were harvested and total RNAs were extracted. cDNAs were synthesized and subjected to qPCR. Relative mRNA levels of Runx2, Ocn and Osx to Gapdh in MSCs were calculated and presented (^#represented comparison between indicated group and negative control; ^#P < 0.05; ^##P < 0.01; *P < 0.05; **P < 0.01)