Figure 1.
Histological appearance and localization of SHH and pFAK Tyr397 in fractured mouse ribs at the early stage after fracturing.
A-P, Photomicrographs show the healing process at 3 (A-H) and 5 (I-P) after fracturing. Sections were stained with HE (A, B, I and J) or for ALP and TRAP (C, D, K, and L), SHH (E, F, M, and N), and pFAK Tyr397 (G, H, O, and P). Q, RT-PCR analysis of tissue samples containing ribs and surrounding tissues within 3 mm from the fracture line on days 1, 3, and 5 after the operation. Each right photo showing a histological section is a magnification of the rectangle-delimited area in the corresponding left photo Bar, 100 µm. Arrowheads: osteoblasts. Arrow: osteoclast. Double arrowheads: migrating bone marrow cells. The data from a typical experiment are presented: similar results were obtained in repeated experiments.
Figure 2.
Histological appearance and localization of SHH and pFAK Tyr397 in fractured mouse ribs at later stages after fracturing.
Photomicrographs show the healing process at 14 (A-H) and 28 (I-P) after fracturing. Sections were stained with HE (A, B, I, and J) or for ALP and TRAP (C, D, K, and L), SHH (E, F, M, and N), pFAK Tyr397 and (G, H, O and P). Each right photo is a magnification of the rectangle-delimited area in the corresponding left photo. Bar, 100 µm. Arrowheads: hypertrophic chondrocytes. Arrows: osteocytes. The data from a typical experiment are presented: similar results were obtained in repeated experiments.
Figure 3.
Effect of SHH on the FAK expression in MC3T3-El cells.
A, RT-PCR analysis of FAK mRNA expression in MC3T3-El cells after treatment with 500 ng/ml SHH. B, Immunoblot analysis of FAK Tyr397 in MC3T3-El cells after incubation with or without 500 ng/ml SHH. The data from a typical experiment are presented: similar results were obtained in repeated experiments.
Figure 4.
Effect of FAK knockdown by short-hairpin FAK RNAs on MC3T3-El cells and on their morphology.
A, Immunoblot analysis of FAK and pFAKTyr397 expression in short-hairpin FAK RNA (shFAK #1-#5)-infected MC3T3-El cells. Control, non-infected MC3T3-E1 cells. Shcontrol, control shRNA-infected group. B, Immunofluorescence staining for pFAK Tyr397 (green) and F-actin (red) and 4’6’-diamidino-2-phenylindole staining (blue) in control, shcontrol and shFAK#5 RNA-infected MC3T3-El cells. Negative control group was treated with antibody dilution buffer minus pFAKTyr397 antibody. The data from a typical experiment are presented; similar results were obtained in repeated experiments.
Figure 5.
Effect of SHH on the proliferation, adhesion, and migration of FAK knockdown MC3T3-El cells.
A, Suppression of growth in shFAK-infected MC3T3-El cells (n = 5). B, Effect of SHH on the proliferation of control or shFAK-infected MC3T3-El cells (n = 5). C, Suppression of cell adhesion of shFAK-infected MC3T3-El cells (n = 5). D, Effect of SHH on the adhesion of control or shFAK-infected MC3T3-El cells (n = 5). E, Suppression of cell migration by shFAK-infected MC3T3-El cells (n = 5). F, Effect of SHH on the migration of control or shFAK-infected MC3T3-El cells after 36 h treatment (n = 5). The data from a typical experiment are presented: similar results were obtained in three separate experiments. Data present mean ± SD. Statistical significance was defined as * P < 0.05 and ** P < 0.01.
Figure 6.
Effect of shFAK and SHH on MC3T3-El cell differentiation.
A, Suppression of ALP staining in shFAK-infected MC3T3-El cells (n = 5). B, Suppression of Alizarin red staining in shFAK-infected MC3T3-El cells (n = 5). C, RT-PCR analysis of osterix (OSX), ALP, Runx2, and osteopontin (OPN) mRNA expression in control and shFAK-infected MC3T3-El cells. D, Effect of SHH on the ALP staining of shFAK-infected MC3T3-El cells (n = 5). The data from a typical experiment are presented: similar results were obtained in three separate experiments. Data present mean ± SD. Statistical significance was defined as * P < 0.05 and ** P < 0.01.
Figure 7.
Suppression of osteoclast formation in PTHrP-stimulated co-culture system comprising control MC3T3-E1 cells or shcontrol- or shFAK-infected MC3T3-El cells and murine CD11b+ bone marrow cells.
A, SHH stimulated an increase in the number of TRAP-positive cells in the presence of 10 nM PTHrP only in co-cultures of control or shcontrol-infected MC3T3-El cells and CD11b+ cells (n = 5). Significant differences between the groups are indicated by the brackets: * P < 0.05 and ** P < 0.01. B and C, RT-PCR analysis of RANKL, OPG and PTHrP expression levels in MC3T3-El cells with or without 10 nM PTHrP or 500 ng/ml SHH. The total RNA was extracted 24 h after the start of treatment. The data from a typical experiment are presented: similar results were obtained in three separate experiments.