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sc-12-0426_sm_SupplFigure1.tif1696KSupplementary Fig. 1. siRNA HTRA1 reduces HTRA1 protein within the ECM of hBMSCs. Representative immunofluorescence images of double staining for HTRA1 (red) and DAPI (blue) in HTRA1 siRNA (HTRA1 (H1)) or control siRNA (siRNA (C)) transfected hBMSCs after 10 days without (non-induced) or with (osteogenic) osteogenic induction. Scale bar = 75μm. Abbreviations: HTRA1, high temperature requirement protease A1; ECM, extracellular matrix; hBMSCs, human bone marrow-derived mesenchymal stem cells; DAPI, 4,6-diamidino-2- phenylindole; siRNA, small interfering ribonucleic acid.
sc-12-0426_sm_SupplFigure2.tif1821KSupplementary Fig. 2. Exogenously added HTRA1 protein enhances hBMSC mineralization during the later stages of osteogenic differentiation only. Representative images of Alizarin red stained day 18 (D18) cultures of non-induced hBMSCs (non-induced) and osteogenic induced hBMSCs previously treated either without (osteogenic) or with proteolytically active HTRA1 (5 μg/ml) for the various times indicated. Data is representative of 3 independent experiments. Scale bar = 2 mm. Abbreviations: HTRA1, high temperature requirement protease A1; hBMSCs, human bone marrow-derived mesenchymal stem cells.
sc-12-0426_sm_SupplFigure3.pdf303KSupplementary Fig. 3. HTRA1 does not degrade collagen type I within the ECM of hBMSCs. Representative immunofluorescence images of COL1A1 (green) and DAPI (blue) in day 14 cultures of non-induced hBMSCs and osteogenic induced hBMSCs treated for 4 days without (untreated) or with 5 μg/ml of active (HTRA1Δmac) or inactive (HTRA1ΔmacSA) HTRA1. Scale bar = 75μm. Abbreviations: HTRA1, high temperature requirement protease A1; hBMSCs, human bone marrow-derived mesenchymal stem cells; COL1A, collagen Type I; DAPI, 4,6- diamidino-2-phenylindole.
sc-12-0426_sm_SupplFigure4.pdf299KSupplementary Fig. 4. Osteogenic differentiation of mASC 3D-spheroids. (A-B): Alizarin red staining of mASC 3D-spheroids at 5 (A) and 9 (B) days post-osteogenic induction. Scale bar = 100 μm (C): Control immunofluorescence staining for HTRA1 and IBSP in mASC spheroids at 5 and 9 days post-osteogenic induction. Representative immunofluorescence images of spheroids stained with mouse IgG (M-IgG; green), rabbit IgG (R-IgG; red) and DAPI (blue). Data is representative of 2 independent experiments. Scale bar = 25 μm. Abbreviations: HTRA1, high temperature requirement protease A1; IBSP, Integrin-binding sialoprotein; mASC, mouse adipose-derived stromal cells; DAPI, 4,6-diamidino-2-phenylindole.
sc-12-0426_sm_SupplFigure5.pdf250KSupplementary Fig. 5. Immunohistochemical staining of HTRA1 in paraffin wax sections of mouse bone. (A-C): Representative micrographs of anti-HTRA1 (brown) stained paraffin wax sections of decalcified mouse femora (representative of n = 3) at 7 (A), 14 (B) and 28 (C) days following osteotomy. HTRA1 was detected in the periosteal layer, p, (A), non-mineralized callus tissue (B) and cuboidal cells localized within areas of active bone regeneration (white arrows) (C). HTRA1 was visualized using horseradish peroxidase-diaminodenzidine and sections counterstained with hematoxylin. Scale bar = 100 μm. Abbreviations: cb, cortical bone; HTRA1, high temperature requirement protease A1.
sc-12-0426_sm_SupplFigure6.pdf201KSupplementary Fig. 6. Control immunofluorescence staining for HTRA1 and IBSP in paraffin wax sections of mouse bone. Representative immunofluorescence images of paraffin wax sections of decalcified mouse femora (representative of n = 3) at 28 days following osteotomy stained with DAPI (blue), mouse IgG (M-IgG; green) or rabbit IgG (R-IgG; red). Scale bar = 50 μm. Abbreviations: HTRA1, high temperature requirement protease A1; IBSP, Integrin-binding sialoprotein; DAPI, 4,6-diamidino-2-phenylindole.
sc-12-0426_sm_SupplTable1.pdf25KSupplemental Table 1.

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