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sc-12-1149_sm_SupplFigure1.TIF2074KFigure S1. Elevated levels of IFN-γ and TNF-α in OVX mice. Immunostaining showed increased IFN-γ and TNF-α positive cells (white arrows) in bone marrow and derma, but not in subcutaneous fat, in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.
sc-12-1149_sm_SupplFigure2.pdf266KFigure S2. The number of dermal MSCs is reduced in OVX mice. Immunohistochemical staining of MSC markers CD105, CD44 and CD73 showed markedly reduced number of dermal MSCs in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.
sc-12-1149_sm_SupplFigure3.TIF1019KFigure S3. OVX results in trabecular bone deterioration and derma atrophy in mice. (A, B) H&E staining showed markedly thinner trabecular bone and reduced dermal thickness, respectively, in OVX mice when compared with sham-operated mice. n = 8 animals per group. *, P < 0.05. Scale bars, 50 μm.
sc-12-1149_sm_SupplFigure4.TIF1905KFigure S4. IFN-γ and TNF-α synergistically cause MSC deficiency in nude mice. (A, B) H&E staining showed that only IFN-γ/TNF-α treatment group showed marked loss of trabecular bone and derma atrophy. n = 8 animals per group. Scale bars, 100 μm.
sc-12-1149_sm_SupplFigure5.TIF1227KFigure S5. Local administration of IFN-γ/TNF-α causes MSC deficiency in nude mice. When IFN-γ and TNF-α, either alone or in combination, were subcutaneously transplanted with hydrogel as a controlled release system to nude mice, only IFN-γ–/TNF-α–infused group showed significantly reduced population doublings (A) and capacity to form mineralized nodules (B) in DMSCs. (C) H&E staining demonstrated that only IFN-γ/TNF-α locally transplanted with hydrogel showed significant derma atrophy. n = 5 animals per group. *, P < 0.05. Scale bars, 100 μm.
sc-12-1149_sm_SupplFigure6.TIF557KFigure S6. IFN-γ and TNF-α impair MSC differentiation via NFκB/SMAD7 pathway. (A) Western blot showed upregulation of p-NFκBp65, p-IκB, SMAD7 and downregulation of p- SMAD1, RUNX2 and ALP in OVX-derived DMSCs. (B) TNF-α treatment showed a dosedependent NFκB activation. (C, D) Alizarin red staining showed that IFN-γ and TNF-α in combination but not alone caused a markedly reduction in mineralized nodule formation in DMSCs, with upregulated p-IκB, SMAD7 and down-regulated p-SMAD1, RUNX2 and ALP. (E, F) With knockdown of NFκB by IKKα siRNA, IFN-γ-/TNF-α-induced upregulation of SMAD7 and downregulation of p-SMAD1, RUNX2 and ALP were abolished, leading to the rescue of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.
sc-12-1149_sm_SupplFigure7.TIF329KFigure S7. IFNGRα and TNFR1 are required for IFN-γ/TNF-α to snergistically induce activation of NFκB. (A) Combined knockdown of IFNGRα and TNFR1 resulted in markedly inhibition of IFN-γ–/TNF-α–induced activation of NFκBp65 and p-IκB when compared with TNFR1 knockdown group. (B) Knockdown of STAT-1 abolished TNF-α-/IFN-γ–induced activation of NFκB pathway. (C, D) Western blot analysis confirmed efficacy of siRNA in inhibiting IKKα, IFNGRα, TNFR1, STAT-1 and SMAD7.
sc-12-1149_sm_SupplFigure8.tif443KFigure S8. SMAD7 knockdown rescues impaired differentiation of BMMSCs and DMSCs. (A) Western blot showed that knockdown of SMAD7 downregulated p-SMAD1, RUNX2 and ALP in BMMSCs without downregulating p-IκB. (B) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in BMMSCs. (C) Western blot showed that knockdown of SMAD7 downregulated RUNX2 and ALP in DMSCs without downregulating p-IκB. (D) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ–/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.
sc-12-1149_sm_SupplFigure9.TIF2332KFigure S9. Long-term IFN-γ/TNF-α exposure increases susceptibility to MSC malignant transformation via NFκB–induced oncogenes. (A) Immunostaining of pan-CK showed negative cells in MCA–induced tumor in OVX mice. (B) Immunostaining of the tumors derived from s.c. inoculation of IFN-γ–/TNF-α–treated BMMSCs showed elevated expressions of VIM and PCNA, but negative expression of pan-CK. (C) Immunostaining of p-NFκB, c-FOS and c- MYC in bone marrow showed more positive cells in aging OVX mice. Scale bars, 50 μm.
sc-12-1149_sm_SupplFigure10.TIF1455KFigure S10. Aspirin rescues osteoporosis and derma atrophy in OVX mice. H&E staining showed that systemically applied aspirin rescued the deteriorated trabecular bone structures (A) and derma atrophy (B) in OVX mice. Scale bars, 100 μm. *, P < 0.05.
sc-12-1149_sm_SupplFigure11.TIF313KFigure S11. Schematic diagram illustrating the mechanism in IFN-γ– and TNF-α–induced MSC deficiency and MSC tumorigenesis. Elevated levels of IFN-γ and TNF-α together, but not independently, synergistically cause MSC deficiency via NFκB–mediated activation of SMAD7, whereas long-term elevated levels of IFN-γ and TNF-α synergistically result in increased tendency toward MSC malignant transformation through NFκB–mediated upregulation of the oncogenes c-Fos and c-Myc.

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