Fig 2: DDR1 promoted mineralization in MC3T3-E1 cells. Knocking down Ddr1 (shDdr1) in ME3T3-E1 cells was performed with (A) mineralization assay staining by Alizarin red and (B) ALP activity. The quantitative results are shown below. (C) The protein levels of DDR1, Runx2, BMP2, ALP, Col-I, OC, and GAPDH in ME3T3-E1 cells were assessed. Quantitation of the results for shDdr1 compared with shLacZ and all other results is shown below. ME3T3-E1 cells overexpressing Ddr1 (ovDdr1) were subjected to (D) the mineralization assay staining by Alizarin red and (E) ALP activity. The quantitative results are shown below. (F) The protein levels of DDR1, Runx2, BMP2, ALP, Col-I, OC, and GAPDH were assessed in ME3T3-E1 cells. Quantitation of ovDdr1 expression compared with shLacZ overexpression is shown below. Each group included n = 3 independent groups; * p = 0.05, ** p = 0.01.

Fig 3: DDR1 regulated osteoblast differentiation through p38 phosphorylation. (A) The protein levels of phosphorylated p38 and ERK in ME3T3-E1 cells with Ddr1 knockdown (shDdr1). The quantitative result was presented as the pp38/p38 and pERK/ERK ratios and normalized with shLacZ. (B) Gene expression of Runx2, ALP, Col-I, OC in ME3T3-E1 cells were treated with the p38 inhibitor. (C) Western blot detected the protein level of phosphorylated p38, Runx2, Col-I, ALP, and OC Runx2 in MC3T3-E1 treated with DMSO (control) and SB203580 (p38 inhibitor). The quantitative results show the ratios of pp38/p38 and Runx2/GAPDH, Col-I/GAPDH, ALP/GAPDH, and OC/GAPDH and were normalized with DMSO. (D) The protein levels of Runx2, pp38, p38 and GAPDH in ME3T3-E1 cells overexpressing Ddr1 (ovDdr1) and treated with SB203580. Quantitation of the results of ovDdr1+DMSO compared with vehicle+DMSO and of ovDdr1+SB203580 compared with vehicle+SB203580 and normalized with those of vehicle+DMSO is shown below. Each group included n = 3; * p = 0.05, # p = 0.05, ** p = 0.01, ## p = 0.01 and * means compare with shLacZ or DMSO or vehicle, and # mean compare with ovDdr1.

Fig 4: Knocking out Ddr1 in osteoblasts caused downregulation of the gene expression of markers related to osteogenesis. (A) Gene expression of Runx2, BMP2, ALP, OC, and Col-I was assessed in calvarial bone of Ddr1f/f-4OHT and OKO?Ddr1 mice. (B) IHC staining of Col-X, MMP13, Runx2, and BMP2 in femurs from 4-week-old Ddr1f/f-4OHT and OKO?Ddr1 mice. Magnifications were 100× and 400× with scale bars of 200 µm and 50 µm, respectively. (C) Quantitation of Col-X, MMP13, Runx2, and BMP2 expression in the trabecular area. Each group n = 6; * p = 0.05, ** p = 0.01.

Fig 5: Generation of osteoblast-specific (a1(I) collagen-CreERT; Ddr1f/f) OKO?Ddr1 mice. The calvarial bone was extracted from Ddr1f/f-4OHT and OKO?Ddr1 mice on postnatal days 4~5, and (A) gene expression of Ddr1 and (B) protein level of DDR1 were detected. The ratio of DDR1/GPADH is expressed relative to the Ddr1f/f-4OHT mice was quantified. Each group n = 6. (C) IHC staining of DDR1 and DDR2 in cortical bone and articular cartilage of femurs from 4-week-old mice; black arrow indicates DDR1-positive cells; black frame indicates the cortical region. (D) Quantitative results by tissue faxes normalized with Ddr1f/f-4OHT. Magnifications of 400X are shown, with scale bars of 50 µm. Each group, n = 6; * p = 0.05, ** p = 0.01.