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1 Supplementry Informtion Non-nonil prevents skeletl ging n inflmmtion y inhiiting NF-κB Bo Yu, Ji Chng, Yunsong Liu, Jiong Li, Kreen Kevork, Khli Al Hezimi, Dn T. Grves, No-Hee Prk, Cun-Yu Wng Supplementry Figures 7 Nture Meiine: oi:.8/nm.86
2 Supplementry Figure N TG TG TG TG TG TG6 TG7 TG8 TG9 N P P BMD (mg m ) -TG Age (months) BV/TV (%) 6 Time (months) Runx mrna 6 MSC/ MSC/ Time (weeks) e Sp7 mrna MSC/ MSC/ Time (weeks) f Isp mrna MSC/ MSC/ Time (weeks) g Bglp mrna MSC/ MSC/ Time (weeks) Supplementry Figure promotes postntl one formtion in vivo. () Southern lot of trnsgene expression in founer mouse lines. ( ) μct nlysis of BMD () nbv/tv() of-, - n -month-ol n mie (TG-). n= mie per group. P <.. ( g) Rel time RT-PCR nlysis of osteogeni mrker genes inluing Runx (), Sp7 (e), Isp (f) nbglp (g) mrna expression in primry one mrrow MSCs isolte from femurs of -month-ol n mie, fter osteogeni inution tretment for inite times. Nture Meiine: oi:.8/nm.86
3 Supplementry Figure Tnf Cox- Supplementry Figure ttenutes the expression of NF-κB-regulte moleules in vivo inue y. ( ) Immunostining of NF-κB-epenent Tnf (), Cox- (), n () surrouning treulr ones in the istl metphysis of n mie two months fter or shm opertion. Sle rs, 6 µm. Nture Meiine: oi:.8/nm.86
4 Supplementry Figure TNFtg TNFtg/ Arthritis sore TNF T/W μct (nkle) Tiiotlr joint e Interigitl joint f Cox- TNFtg TNFtg/ g TNFtg TNFtg/ Supplementry Figure llevites rthritis inue y TNF. ( ) Photogrphs of hinpws n nkle joints () showing swelling (yellow rrow) s well s μct reonstrution of nkle n tiiotlr joints () showing ony erosions (re rrow) from -month-ol, TNFtg n TNFtg/ mie. Averge rthritis sores () were given se on the egree of swelling n joint evition. n = 8 hinpws for n TNFtg/ groups; n = hinpws for TNFtg group. P <.. ( e) H&E stining of tiiotlr () n interigitl (e) joints showing joint rtilge estrution n one erosions ue to invsion of inflmmtory ells (lk rrows). (f,g) Immunostining of NF-κB-epenent Cox- (f) n (g) in istl femorl metphysis of -month-ol,, TNFtg n TNFtg/ mie. Sle r, mm (); μm ( e); μm (f g). Nture Meiine: oi:.8/nm.86
5 Supplementry Fig. 8 months 8 months β-tin β-tin -month-ol -month-ol months Tnf 8 months Cox- Supplementry Figure ttenute the expression of NF-κB-regulte moleules in ging mie. () RT-PCR of enogenous n trnsgeni mrna expression in mie n mie of vrious ges. () RT-PCR of trnsgene mrna expression in mie n mie of vrious ges. () Immunostining of proteins in young (-month-ol) n ge (8-month-ol) n mie. () Immunostining of NF-κB-epenent Tnf, Cox- n in istl metphysis of - month-ol n mie. Sle rs, μm( ). Nture Meiine: oi:.8/nm.86
6 Supplementry Fig. + e f g Trp mrna Il6 mrna Il6 mrna Tnf mrna Trp mrna Bir mrna Cox mrna # OC/Totl ells mrna mrna Bir mrna h Ctsk mrna Ctsk mrna p-p8 p8 p-jnk Jnk p-erk Erk α-tuulin # OC/ Totl ells... + min Supplementry Figure. iretly inhiits osteolst ifferentition inue y. ( ) TRAP stining showing osteolst formtion from one mrrow mrophges () n RAW6.7 ells () inue y or with. (,) Rel time RT-PCR of Trp, n Ctsk mrna in one mrrow mrophges () n RAW6.7 ells (). (e,f) Rel time RT-PCR of Il6 n Bir in one mrrow mrophges n RAW6.7 ells (f). (g) Rel time RT-PCR of Tnf n Cox- in one mrrow mrophges. (h) Immunolots showing the phosphoryltion of p8, Jnk, n Erk of lystes from one mrrow mrophges stimulte with, or with. Sle rs, μm ( ). P<.; P <.. Nture Meiine: oi:.8/nm.86
7 Supplementry Figure BMD (mg m ) TRAP i Serum Trp (U L ) O.N/BS (μm ) BFR (μm μm y ) f g h + p6 Tnf Cox- j + 8 p6 IOD(x) HE Serum Il-6 (pg ml ) Serum Tnf (pg ml ) BV/TV (%) e O.S/BS 8 Supplementry Figure 6 r prevents osteoporoti one loss y inhiiting NF-κB. ( ) μct reonstrution (), BMD () n BV/TV () of istl femorl metphysis regions from mie fter shm opertion, n immeitely followe y r injetion. () BFR mesurement from ul lein leling of mie. (e f) H&E stining (e) n TRAP stining (f) in istl metphysis of mie. (g) Morphometri nlysis of osteolst ounts in istl femorl metphysis. (h) ELISA of Trp onentrtions in serum. (i) Immunostining showing tive p6, Tnf, Cox- n in istl femorl metphysis. (j) ELISA of serum onentrtions of Tnf n Il-6. For,,, n g j, n = 8 mie for shm group; n = mie per group for mie reeiving n with preventive r injetion. P <., P <., unpire two-tile t-test. Sle rs, μm (); μm (e); μm (f) n (i). Nture Meiine: oi:.8/nm.86
8 Supplementry Fig. 7 + Tnf Cox- Supplementry Figure 7 r proteins ttenute tivtion of NF-κB-epenent moleules inue y. Immunostining of NF-κB-epenent Tnf, Cox- n in istl metphysis of mie. Sle rs, μm. Nture Meiine: oi:.8/nm.86
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