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jbmr1992-sm-0001-SuppFig-S1.tif16520KSupplemental Figure 1. (A) Expression levels of TβRI, TβRII, and β-actin were detected by western blot in cell lysates collected from WT, Nf1+/−, and Nf1−/− osteoblast progenitors. (B) Western blot showing p-Smad2, Smad2, and β-actin in MSCs following 2 min stimulation with TGF-β1 in the presence or absence of SD-208 (100 nM). The bar graph above represents the fold change in p-Smad2 compared to the protein loading control. (C) WT and Nf1−/− MSCs were cultured in osteogenic differentiation medium supplemented with TGF-β1 in the presence or absence of SD-208. Representative photomicrographs (left panel) show ALP positive osteoblasts (×100 magnification). The bar graph (right panel) shows the fold change in ALP activity relative to the WT control. n = 4. **P < 0.01, ***P < 0.0001. (D) 5 × 104 WT and Nf1−/− MSCs were seeded in 12-well plates and cultured in the presence of TGF-β1 (1 ng/mL) with or without SD-208 (1 µM) for 7 days. Proliferation was quantified as the fold change in cell number relative to the input as shown. n = 3. **P < 0.01, ***P < 0.0001. (E) The expression pattern of CD45, CD105, and CD29 cell surface antigens in WT and Nf1−/− MSCs was compared by flow cytometry as shown in the representative histograms.
jbmr1992-sm-0001-SuppFig-S2.tif5376KSupplemental Figure 2. (A) TGF-β1 mRNA expression was measured in WT MSCs following retroviral transduction with control vector (MSCV-pac) versus the functional, full length NF1 GRD construct. n = 3 technical replicates. The experiment was repeated twice independent with similar results. No significant difference was observed when comparing Tgfb1 gene expression between WT MSCs traduced with either the MSCV-pac or MSCV-NF1 GRD-pac constructs. (B) p-Smad2, Smad2, and β-actin levels were detected by western blot in MSCs stimulated with TGF-β1 following transduction with either MSCV-pac or MSCV-NF1 GRD retroviral vectors. This assay was repeated twice with similar results. The fold change in p-Smad2 was determined relative to the level of total Smad2 protein as shown. We observed no change when comparing WT MSCs transduced with MSCV-pac versus the MSCV-NF1 GRD-pac construct.
jbmr1992-sm-0001-SuppFig-S3.tif12671KSupplemental Figure 3. (A) Bone marrow mononuclear cells isolated from WT and Nf1+/− mice were cultured in medium supplemented with M-CSF and RANKL in the presence or absence of TGF-β1 (1 ng/mL). SD-208 was added in increasing doses of 100 nM, 500 nM, and 1000 nM. After 6 days culture, adherent osteoclasts were fixed and stained with Alex Flour® 488 labeled phalloidin (green) to visualize osteoclast actin rings and Hoeschst (blue) to label nuclei. Representative ×200 photomicrographs are shown above. (B) Osteoclasts cultured from the peripheral blood of a NF1 patient and a matched, healthy control following TGF-β1 stimulation and or SD-208 inhibitor treatment. **P < 0.01, ***P < 0.001.
jbmr1992-sm-0001-SuppFig-S4.tif20914KSupplemental Figure 4. TGF-β signaling potentiates osteolytic activity in Nf1+/− mice following OVX proresorptive challenge. (A) The percentage change in bone mineral density (BMD) was determined by pDEXA measurements before and after 6 wks treatment. n = 10-12. *P < 0.05, ***P < 0.001. (B) Representative micro-computed tomography (µCT) shows femoral trabecular bone in Sham and OVX mice following 6 wks treatment with vehicle (1% methylcellulose) or TβRI inhibitor SD-208 (60 mg/kg/d). (C) Trabecular bone volume fraction (BV/TV) was quantified in the distal femur by µCT. n = 10-12. *P < 0.05, ***P < 0.001. Trabecular microarchitecture parameters including connectivity density (Conn.D.) (D), trabecular number (Tb.N) (E), trabecular spacing (Tb.Sp) (F), and structure model index (SMI) (G) were quantified by µCT in OVX mice receiving SD-208 treatment as compared to controls. n = 10-12. *P < 0.05, **P < 0.01, ***P < 0.001. (H) Representative longitudinal sections of hematoxylin and eosin (H&E) stained femora, ×25 magnification. (I) Osteoclast numbers on the bone surface (Oc.N/BS) were counted manually on tartrate resistance acid phosphatase (TRACP) stained sections at ×200 magnification. n = 10-12. *P < 0.05, ***P < 0.001. (J) Serum levels of the C-terminal cross-linking telopeptide of type I collagen (CTX) were measured by ELISA. n = 10-12. *P < 0.05, **P < 0.01.
jbmr1992-sm-0001-SuppFig-S5.tif15111KSupplemental Figure 5. (A) Representative longitudinal sections of trichrome stained femora (×25 magnification) following 4 wks treatment with SD-208, 60 mg/kg/d, versus vehicle control. Trabecular microarchitecture parameters including (B) connectivity density (Conn. D.), (C) trabecular number (Tb.N), and (D) trabecular spacing (Tb.Sp) were quantified by µCT in femora following 4 wks treatment with SD-208, 60 mg/kg/d, as compared to vehicle control. n = 7-12. *P < 0.05, **P < 0.01, ***P < 0.001.
jbmr1992-sm-0001-SuppFig-S6.tif13899KSupplemental Figure 6. (A) Representative photomicrographs show TRACP stained sections of the distal femur at ×25 and ×100 magnification, respectively. (B) The number of osteoclasts normalized to the trabecular bone surface (Oc.N/BS) were counted manually at ×200 magnification. n = 4 mice per group. **P < 0.01.
jbmr1992-sm-0001-SuppFig-S7.tif6332KSupplemental Figure 7. Radiographs showing tibial fracture healing in WT controls and Nf1flox/−;Col2.3Cre mice treated with either vehicle or SD-208, 60 mg/kg/d, for 6 wks.
jbmr1992-sm-0001-SuppFig-S8.tif8603KSupplemental Figure 8. (A) Gelatinase activity of MMP-2 and MMP-9 in WT versus Nf1+/− myeloid cell conditioned medium were quantified by densitometry as shown. n = 3. **P < 0.01. (B) The activity of MMP-2 and -9 in the serum of a NF1 patient (NF1) and a matched, healthy control was determined by zymography. (C) The bar graph represents the fold change in gelatinse activity of MMP-2 in the serum of the NF1 patient relative to the healthy control shown in panel B.

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