Fig 1: rComp mediated the effects of ADAMTS7 on OS cell proliferation, migration, invasion and osteogenic differentiation. (A) A CCK‐8 assay was conducted after 48 h to detect the proliferation of ADAMTS7 overexpressing MG63 cells with or without rComp (100 ng·mL−1). Values are expressed as the mean ± SEM from three independent experiments performed in duplicate. (B) A transwell assay was performed to detect the migration and invasion ability of ADAMTS7 overexpressing MG63 cells under rComp treatment (100 ng·mL−1). Scale bar = 200 μm. The number of migrated cells (C) and invaded cells (D) was calculated and quantified as the mean ± SEM for three independent experiments.*P < 0.05. NS, not significant. (E) Biomineralizations were measured in MG63 cells to evaluate the effect of rComp on the osteogenic differentiation triggered by ADAMTS7. Calcium deposition was analyzed and quantified as the mean ± SEM (n = 3). *P < 0.05. NS, not significant. Data in (A), (C), (D) and (E) were analyzed by one‐way ANOVA with the Student–Newman–Keuls test for multiple comparisons. (F) Representative images of co‐immunoprecipitation for Comp and BMP2. IB, immunoblot. (G) Representative images for western blotting of osteogenic markers in MG63 cells with or without rComp (100 ng·mL−1) pretreatment. The relative expression of BMP2, Runx2 and p‐Smad1/5 was quantified as the mean ± SEM for three independent experiments. **P < 0.01, *P < 0.05 by two‐way ANOVA with Bonferroni's test.
Fig 2: ADAMTS7 was down‐regulated in OS tissues. (A) The relative mRNA expression of ADAMTS7 in OS compared to the corresponding adjacent tissues (n = 49). (B) The results are presented as the log2(fold‐change) in OS samples relative to the adjacent tissues. (C) The relative mRNA expression of ADAMTS7 in poor‐ (n = 28) and well/moderately differentiated (n = 21) OS tissues. (D) Survival curve for high‐ and low‐ADAMTS7‐expression OS patients. P value by log‐rank (Mantel–Cox) test. (E) Representative images of western blotting in OS and adjacent tissues (n = 26). The relative expression level of ADAMTS7 was quantified. (F) Representative images of IHC in OS and adjacent tissues (200×). Scale bar = 20 μm. Data in (A), (B), (C) and (E) were analyzed by Student's t‐test.
Fig 3: Protein expression levels of ADAMTS-7 in the aneurysm aorta. (A) Aneurysm aorta and (B) control aorta representative images and (C) quantification of protein ADAMTS-7 expression levels detected via immunohistochemistry. ADAMTS-7 was stained as brown chromogen which was primarily located in the media and along the intima-medial border. Semi-quantitative analysis demonstrated increased expression of ADAMTS-7 in aneurysm aortas (n=4) compared with the control aortas (n=6). *P<0.05 vs. control. Magnification, ×400. ADAMTS-7, a disintegrin and metalloproteinase with thrombospondin motifs 7.
Fig 4: Silence of ADAMTS7 promoted the cancerous ability of OS cells. (A, B) Proliferation of OS cell lines MG63 and SAOS2 at 24, 48 and 72 h was determined by CCK‐8 after transfection with siRNA. Data are expressed as the mean ± SEM from three independent experiments performed in duplicate. *P < 0.05. (C, D) Cell‐cycle distribution AT 24 h was confirmed by propidium iodide staining and FACS analysis in MG63 and SAOS2 cells. Data are expressed as the mean ± SEM from four independent experiments. *P < 0.05. (E, F) Representative images of cell migration 24 h after scratching (magnification 100×). Scale bar = 1 mm. The mean distances migrated by cells at 6, 12 and 24 h were quantified. Data are expressed as the mean ± SEM from three independent experiments. *P < 0.05. Data in (A) to (F) were analyzed by two‐way ANOVA with Bonferroni's test for multiple comparisons. (G, H) Representative images for a transwell assay in MG63 and SAOS2 cells (magnification 100×). Scale bar = 200 μm. Upper: Migration. Lower: Invasion. The numbers of transmembrane cells were calculated and quantified. Data are expressed as the mean ± SEM (n = 3). ***P < 0.001, *P < 0.05 by two‐tailed Student's t‐test.
Fig 5: ADAMTS7 reinforced osteogenic differentiation and suppressed OS pathogenesis via BMP2. (A, B) Representative images of biomineralization by alizarin red staining in MG63 cells stimulated by ascorbic acid and β‐glycerophosphate for 21 days with ADAMTS7 overexpressing (A) or silencing (B). Calcium deposition was analyzed and quantified as the mean ± SEM (n = 3). *P < 0.05 two‐tailed Student's t‐test. (C, D) RT‐PCR analysis in different treated OS cells revealed altered mRNA expression of genes involved in osteoblast differentiation. Data are expressed as the mean ± SEM from three independent experiments performed in duplicate. *P < 0.05 compared to the pcDNA3.1 group. # P < 0.05 compared to the siRNAscrmable group. (E) Representative images for western blotting of osteogenic protein in transfected OS cells. Left: MG63 cells. Right: SAOS2 cells. The relative expression of BMP2, Runx2 and p‐Smad1/5 was quantified as the mean ± SEM for three independent experiments. *P < 0.05 compared to the pcDNA3.1 group. # P < 0.05 compared to the siRNAscrmable group. Data in (C) to (E) were analyzed by two‐way ANOVA with Bonferroni's test for multiple comparisons. (F) Correlation of ADAMTS7 and BMP2 protein in OS tissues. (G) Proliferation and (H) migration ability were assessed in ADAMTS7 overexpressing cells with or without Noggin (100 ng·mL−1) stimulation. Data are expressed as the mean ± SEM (n = 3). *P < 0.05. NS, not significant by one‐way ANOVA followed by the Student–Newman–Keuls test for post‐hoc comparison.
Supplier Page from Abcam for Anti-ADAMTS7 antibody