ΟǵBuffer composition (1) Sample buffer (5X)Ǻ
7.81 ml stacking buffer (0.5 mM Tris-HCl, pH=6.8) 2.5 g SDS
14.36 ml glycerol
6.25 ml ȕ-mecaptoethanol 2 mg bromophenol blu уΒԛНԿ 50 ml
0.05 ml 10 ʘ ammonium persulfate 0.005 ml TEMED
8 ʘ Stacking gel (5 ml)Ǻ 11.5 ml H2O
6.7 ml 30 ʘ acrylamide mix 6.3 ml 1.0 M Tris (pH=6.8) 0.025 ml 10 ʘ SDS
0.025 ml 10 ʘ ammonium persulfate 0.015 ml TEMED
0.25 ml 10 ʘ ammonium persulfate 0.01 ml TEMED
(3) EMSA-6 ʘ non-denaturing polyacrylamide gel 21.5 ml H2O
6 ml 30 ʘ acrylamide mix 3 ml 5X TBE
0.3 ml 10 ʘ ammonium persulfate 0.015 ml TEMED
(4) RIPA buffer 1 ml lysis buffer 50 mM PMSF 50 mM Na3Vo4
42
10ȝg/ml Aprotinin 1 mM NaF
(5) Running buffer (10X) 30.2 g Tris-base
140 g Glycine 10 g SDS
уΒԛНԿ 1 L
(6) Transfer buffer (10X) 30 g Tris
144 g Glycine уΒԛНԿ 1 L
(7) Lysis buffer 50 mM Tris pH7.5 150 mM NaCl 10 mM EDTA 1 ʘ NP-40
0.25 ʘ deoxycholate
(8) TBS-T (20X) 48.46 g Tris 0.5 M EDTA
58.44 g NaCl (pH=7.5) уΒԛНԿ 1 L
Ѥǵीϩ!
ҁჴᡍ่݀а sigmaplot ी೬ᡏՉϩǶঁჴᡍ֡ՉΟԛа
ᐱҥ၂ᡍǴჴᡍ่݀а mean ± S.E. ߄ҢǴа student’s t test ՉኧᏵϩ
Ǵpɦ0.05 ܭी߄ҢԖᡉৡ౦Ƕ
44
ಃѤകġ ่݀
ǵ౦Ч✉़ғނჹܭମᡎ୷፦༸ಒझϷ RAW264.7 ѮᏘಒझޑಒझ Ӹࢲޑቹៜ
२ӃǴࣁΑዴۓ౦Ч✉़ғނǴჹܭମᡎ୷፦༸ಒझϷ RAW264.7 ѮᏘಒझࢂցڀԖࢥ܄ǴӢԜǴӧಒझύуΕ౦Ч✉़ғނ (5 ȝM) ٿ ϺࡕǴךॺճҔ MTT ٰෳໆಒझӸࢲǶҗჴᡍύวǴ౦Ч✉़ғ ނǴჹܭମᡎ୷፦༸ಒझ܈ RAW264.7 ѮᏘಒझޑಒझӸࢲؒԖቹៜ (Fig. 8A, B)Ƕ
Βǵ౦౦Ч✉़ғނڋᇑମಒझԋ
ᇑମಒझࢂߐޑൂਡಒझ0ѮᏘಒझৎޑԋǴҗମᡎՈᖿ ނ܌ϩϯԶԋ (bone marrow hematopoietic precursors)Ƕךॺҗԋԃ܄
SDႵύڗрߏମǴ٠ϩᚆрମᡎ୷፦༸ಒझǴᅿ1×106ಒझܭ24ϾዬύǶ ӧᇑମಒझ០ނମᡎ୷፦༸ಒझύǴ๏ϒRANKL (50 ng/ml) аϷ M-CSF (20 ng/ml) 5ϺǴаᇨᏤԋዕޑᇑମಒझԋǴԶԋዕޑᇑମಒझ ϣԖӭਡޑቻǴӢԜךॺёаճҔTRAPѐࢉਡǴεܭ3ᗭਡࣁᇑମಒ झǶ
๏ϒRANKLکM-CSFԖᡉޑᇑମಒझғԋǴՠࢂӝٳ๏ϒ౦Ч
✉़ғނǴܴᡉڋΑᇑମಒझޑԋ (Fig. 9A)ǶFig. 9BࣁໆϯკǶ
ӕ ኬ ޑ ݩ Ψ ӧ RAW264.7 Ѯ Ꮨ ಒ झ ύ ё а ࣮ ډ Ƕ ӧ 2×104ᗭ RAW264.7ѮᏘಒझύǴ๏ϒRANKL (50 ng/ml) аϷM-CSF (20 ng/ml)Ǵ
܈ӝٳ๏ϒ౦Ч✉़ғނǴ5Ϻࡕёа࣮ډᇑମಒझޑғԋǴՠӝٳ๏
ϒ౦Ч✉़ғނǴڋᇑମಒझޑԋ!(Fig. 9C)Ƕ
ӢࣁԋዕޑᇑମಒझύǴ֖ԖĮvȕ3 integrin (Nesbitt et al., 1993)Ǵ܌
аךॺёаෳໆĮvȕ3բᇑମಒझԋዕޑǶӧRAW264.7ѮᏘಒझ ύǴ๏ϒRANKL (50 ng/ml) аϷM-CSF (20 ng/ml)Ǵ܈ӝٳ๏ϒ౦Ч
✉़ғނǴ5ϺࡕǴճҔࢬԄಒझሺෳໆĮvȕ3߄ໆǶ๏ϒRANKLаϷ M-CSFǴᡉቚуĮvȕ3߄ໆǶ࣬ၨܭRANKLǴӝٳ๏ϒ౦Ч✉़ғ ނǴܴᡉ෧ϿĮvȕ3߄ໆ (Fig. 9D)Ƕ
җаޑჴᡍ่݀ᡉҢǴ౦Ч✉़ғނǴёаڋᇑମಒझޑ
ԋǶ!
Οǵ౦Ч✉़ғނڋ RANKL ܌ᇨᏤޑ ERKǵp38 аϷ JNK ࢲ܄
MAPKs (mitogen-activated protein kinase) х֖3ঁԋǴERKǵp38 аϷJNKǴԖࣴزࡰрǴѬॺکRANKL܌ፓޑᇑମಒझԋԖᜢ (Tsai et al., 2008)ˤࣁΑှញ౦Ч✉़ғނޑૻ৲ሀၡ৩ǴךॺճҔՋБᗺ ᏀݤෳໆMAPKsޑࢲ܄Ƕ!
ӧRAW264.7ѮᏘಒझύǴ๏ϒ܈ό๏JFL-142 (0.3 ȝM) ϷJFL-144 (0.3ȝM)Ǵ30ϩដࡕǴ๏ϒRANKL (50 ng/ml)Ǵ60ϩដࡕǴԏಒझǴ٠
46 (Bossard et al., 1999)Ǵ܌аךॺճҔՋБᗺᏀݤаϷ DNA ᆶೈқ่ӝૈ
ΚϩݤǴෳໆ NF-țB ࢲ܄Ƕ
ӧ RAW264.7 ѮᏘಒझύǴ๏ϒ܈ό๏ JFL-142 (0.3 ȝM) Ϸ JFL-144 (0.3 ȝM)Ǵ30 ϩដࡕǴ๏ϒ RANKL (50 ng/ml)Ǵ60 ϩដࡕǴԏಒझǴ ٠ճҔ phospho-IKKĮ/ȕ לᡏǵphospho-IțBĮ לᡏϷ phospho-p65Ser536לᡏ
խࣝᗺᏀǶךॺว RANKL ᡉቚу IKKĮ/ȕˣIțBĮ Ϸ p65Ser536ᕗ ለϯǴՠӝٳ๏ϒ౦Ч✉़ғނǴڋΑ RANKL ܌ᇨᏤቚуޑ IKKĮ/ȕˣIțBĮ Ϸ p65Ser536ࢲ܄ (Fig. 11A)Ƕ
ௗǴӧ RAW264.7 ѮᏘಒझύǴ๏ϒ܈ό๏ JFL-142 (0.3 ȝM) Ϸ JFL-144 (0.3 ȝM)Ǵ30 ϩដࡕǴ๏ϒ RANKL (50 ng/ml)Ǵ120 ϩដࡕǴԏ
ಒझǴ٠ճҔ EMSA ෳໆ DNA ᆶೈқ่ӝૈΚǶךॺว RANKL
ᡉቚу NF-țB ࢲ܄Ǵՠӝٳ๏ϒ౦Ч✉़ғނǴڋΑ RANKL
܌ᇨᏤቚуޑ NF-țB ࢲ܄ (Fig. 11B)Ƕ
ϖǵ౦Ч✉़ғނڋ RANKL ܌ᇨᏤޑ NF-țB ࢲ܄
ࣁΑჴ NF-țB ࢲ܄Ǵୖᆶ౦Ч✉़ғނޑբҔύǴ܌а ךॺճҔ țB հӀሇનࢲ܄ෳۓǴෳໆ NF-țB ࢲ܄Ƕ
ӧ RAW264.7 ѮᏘಒझύǴᙯࢉ țB promoter-luciferaseǴ1 ϺࡕǴ๏
ϒ܈ό๏౦Ч✉़ғނǴ30 ϩដࡕǴ๏ϒ RANKL (50 ng/ml) 24 λਔǴ ԏಒझǴ٠аհӀෳۓሺෳໆǶךॺวǴ๏ϒ RANKL ᡉቚу NF-țB ࢲ܄Ǵ࣬ၨܭ RANKLǴӝٳ๏ϒ౦Ч✉़ғނǴܴᡉڋΑ RANKL
܌ᇨᏤቚуޑ NF-țB ࢲ܄ (Fig. 12)Ƕ
೭٤่݀ᡉҢǴERKǵp38ǵJNK аϷ NF-țB ૻ৲ሀၡ৩Ǵࢂ౦Ч
✉़ғނ෧ϿѮᏘಒझϩϯԋᇑମಒझޑѸाၡ৩Ƕ
!
Ϥǵ౦Ч✉़ғނჹܭମಒझޑಒझӸࢲϐቹៜ
வय़ޑჴᡍளޕǴ౦Ч✉़ғނჹܭᇑମಒझޑԋԖڋբ ҔǴௗགྷाޕၰ౦Ч✉़ғނǴࢂցჹܭମಒझԖቹៜǴ܌а
аΠޑෳ၂Ƕ!
२ӃǴࣁΑዴۓ౦Ч✉़ғނǴჹܭମಒझࢂؒԖࢥ܄ޑǴӢ ԜǴӧ MG-63 cells Ϸ 3T3 cells ύ๏ϒ౦Ч✉़ғނ!(5 ȝM) 2 ϺࡕǴ ךॺճҔ MTT ٰෳໆಒझӸࢲǶҗჴᡍύวǴ౦Ч✉़ғނǴჹ ܭ MG-63 cells ܈ 3T3 cells ޑಒझӸࢲࢂؒԖቹៜޑ (Fig. 13)Ƕ
48
Ύǵ౦Ч✉़ғނ٤ቹៜମಒझޑϩϯբҔ (ALP)
ӢࣁମಒझϩϯࣁԋዕମಒझਔǴϩݜ٤ӢηǴٯӵǴ ALPǵBMP-2ǵOCаϷOPNǴӢԜךॺёаෳໆ೭٤ӢηǴբମಒ झϩϯԋዕޑ (Lee et al., 1999)Ƕ
ӧMG-63ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙
ᙹϸᔈෳໆALPࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭMG-63ಒझύǴ ALPࢲ܄ؒԖቹៜ (Fig. 14A)Ƕ
ӧ3T3ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙ᙹ ϸᔈෳໆALPࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭ3T3ಒझύǴALPࢲ
܄Ԗ٤ቚу (Fig. 14B)Ƕ
Ζǵ౦Ч✉़ғނ٤ቹៜମಒझޑϩϯբҔ (BMP-2)!
ӧMG-63ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙
ᙹϸᔈෳໆBMP-2ࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭMG-63ಒझ ύǴBMP-2ࢲ܄Ԗ٤ቚу (Fig. 15A)Ƕ
ӧ3T3ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙ᙹ ϸᔈෳໆBMP-2ࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭ3T3ಒझύǴBMP-2 ࢲ܄Ԗั༾ቚу (Fig. 15B)Ƕ
ΐǵ౦Ч✉़ғނ٤ቹៜମಒझޑϩϯբҔ (OC)!
ӧMG-63ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙
ᙹϸᔈෳໆOCࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭMG-63ಒझύǴ OCࢲ܄Ԗ٤ቚу (Fig. 16A)Ƕ
ӧ3T3ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙ᙹ ϸᔈෳໆOCࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭ3T3ಒझύǴOCࢲ܄
Ԗั༾ቚу (Fig. 16B)Ƕ
!
Μǵ౦Ч✉़ғނ٤ቹៜମಒझޑϩϯբҔ (OPN)!
ӧMG-63ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙
ᙹϸᔈෳໆOPNࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭMG-63ಒझύǴ OPNࢲ܄ؒԖቹៜ (Fig. 17A)Ƕ
ӧ3T3ಒझύǴуΕ౦Ч✉़ғނ24λਔࡕǴճҔջਔᆫӝ䁙ᙹ ϸᔈෳໆOPNࢲ܄Ƕךॺว౦Ч✉़ғނǴჹܭ3T3ಒझύǴOPNࢲ
܄Ԗั༾ቚу (Fig. 17B)Ƕ
ӢԜǴךॺҗа4ঁମಒझϩϯԋዕޑёаޕၰǴ౦Ч✉
़ғނั༾ቹៜମಒझޑϩϯբҔǶ
ΜǵӧռணϪନႵύ౦Ч✉़ғނૈڋମ፦ࢬѨ
ӧin vitroჴᡍύǴךॺςளޕ౦Ч✉़ғނǴڋᇑମಒझޑ
ԋǴӆٰךॺགྷाޕၰǴӧin vivoჴᡍύǴࢂցΨૈளډ࣬ӕ่݀Ƕ
50
ӢԜךॺճҔሟ܄BALB/cλႵǴռணϪନЋೌǴϩԋ5ಔǴ໒ΘόϪ ନռணǵϪନռண (ovariectomized)ǵаϷϪନռணࡕ๏ϒipriflavoneǵ JFL-142܈JFL-144 (100 ȝg/kg)Ǵ๏ᛰಔճҔဎݙ (i.p.) ޑБԄǴ2 Ϻ๏ᛰԛǴӅ๏3ຼǶϐࡕճҔDEXAෳໆBMCаϷBMDǶ
ךॺวǴϪନռணಔ࣬ၨܭ໒ΘόϪନռணǴᡉၨեޑBMCॶǴ Զ࣬ၨܭϪନռணಔǴ๏ϒipriflavoneǵJFL-142܈JFL-144ǴԖᡉቚу ޑBMCॶ (Fig. 18A*Ƕ!
ԶЪǴϪନռணಔ࣬ၨܭ໒ΘόϪନռணǴΨԖᡉၨեޑBMDॶǴ Զ࣬ၨܭϪନռணಔǴ๏ϒipriflavoneǵJFL-142܈JFL-144ǴԖᡉቚу ޑBMDॶ (Fig. 18B*Ƕ
ӢԜךॺளډکin vitroჴᡍ࣬ӕޑ่݀Ƕ
!
!
ಃϖകġ ፕ
ମ፦ࢬѨࢂ࣬ᜢܭԴϯǵᜢݹǵᕎੱаϷᄌ܄วݹ੯ੰޑЬाୢ
ᚒϐǴऩाᇙрཥޑᛰࠔڋମ፦ࢬѨǴٗሶ൩ሡाݙཀǴӼӄǵ ԖਏǵॄᏼளଆǴ೭൳ঁाᗺࢂख़ाޑ!(Sung et al., 2009)Ƕ౦Ч✉ࢂ
җरጴϩᚆрٰޑϺฅ౦✉़ғނǴ٠මԖࣴزࡰрǴ౦Ч✉ڀԖ לวݹޑࢲ܄ǶԶ౦Ч✉ςӧѱय़ёа࣮ளډǴՠࢂѬѸਔதܺ
ҔǴ٠Ԗ٤ޑୋբҔǶҗӃࣴزࡰрǴ౦Ч✉คݤႣٛମ፦ࢬѨ (Alexandersen et al., 2001)Ƕ܌аךॺӝԋΑཥޑ౦Ч✉़ғނǴࣴزࢂ
ցૈԖ׳ӳޑᕍਏǶӢԜǴҁጇޑࣴزҞޑ൩ࢂགྷाǴ౦Ч✉
़ғނჹܭRANKLᇨᏤᇑମಒझԋޑቹៜǶԶךॺҗମᡎ୷፦༸ಒझ аϷѮᏘಒझύวǴ౦Ч✉़ғނૈڋᇑମಒझޑԋǶԶ RANKL܌ᇨᏤޑERKǵp38ǵJNKаϷNF-țBࢲ܄ǴΨ౦Ч✉़ғ ނڋǶ౦Ч✉़ғނΨૈႣٛռணϪନ܌ᇨวޑମ፦ࢬѨຝǶ!
ࣁΑຑ౦Ч✉़ғނჹܭᇑମಒझԋޑቹៜǴךॺճҔ଼ந ε Ⴕ Ǵ ମ ᡎ ୷ ፦ ༸ ಒ झ Ꭶ س Ǵ а Ϸ Ѯ Ꮨ ಒ झin vitro ๏ ϒ RANKL/M-CSFڈᐟౢғᇑମಒझǶךॺޑ่݀ᡉҢǴ౦Ч✉़ғނૈ
ڋᇑମಒझϩϯǶ
MAPKৎԋࢂಢለ!(proline) ᆅޑํለ (serine)/ ♏
52
ለ (threonine) ᐟ䁙ǴѬӧಒझғߏǵϩϯаϷಒझΫǴתᄽࡐख़ा
ޑفՅ (Hirai et al., 1983)ǶԖ٤Ѧӧޑڈᐟࢲϯ♏ለϷႀለ (tyrosine) ޑᕗለϯǶԶMAPKৎԋхࡴERKǵp38аϷJNK (Berg et al., 2003; Ducy et al., 2000)ǶԶЪԖ٤ࣴزࡰрRANKL/RANK่ӝЇଆ ERKǵp38аϷJNKޑᕗለϯǴԶ೭٤ᕗለϯᏤठᇑମಒझޑϩϯ(Berg et al., 2003; Ducy et al., 2000; Yasuda et al., 1998)ǶӧҁጇࣴزǴךॺճҔ ӕ፦ᕷဂޑRAW264.7ಒझǴܴ౦Ч✉़ғނჹܭᖿᇑମಒझ ځфૈࢂҗRANKLᇨᏤNF-țBࢲ܄܌ፓޑ (Bossard et al., 1999)ǶӢ ԜǴӵ݀ڋNF-țBࢲ܄Ǵ܈ૈቹៜᇑମಒझࢲ܄ǴԶݯᕍମ፦౧
ੱǶӧҁጇࣴزǴךॺჴ౦Ч✉़ғނǴӧRAW 264.7ಒझύǴᙖ
җڋIKKĮ/ȕǵIțBĮϷp65ޑᕗለϯǴܴڋRANKL܌ᇨᏤޑNF-țB ࢲ܄ǶௗճҔᙯࢉțB-luciferaseࡰǴෳໆNF-țBࢲ܄ǴךॺΨว
RANKL܌ቚуޑNF-țBࢲ܄౦Ч✉़ғނ܌ڋǶҗа่݀ё аଷǴ౦Ч✉़ғނӧѮᏘಒझύǴڋNF-țBࢲ܄ǴԶԋᇑମ ಒझԋаϷځӧ֎ԏբҔޑࢲ܄ڋǶ
ମᓐࢂঁፄᚇޑಔᙃǴҗ൳ᅿᜪޑಒझಔԋǴ٠Ъೱុόᘐޑ
ᐕ׳ཥаϷঅൺၸำǶମޑӆ֎ԏϷԋόڐፓǴЪମϩှεܭମ
ԋਔǴёૈԋମ፦౧ੱ (Tsai et al., 2008)Ƕךॺӧҁጇࣴزว
Ǵ౦Ч✉़ғނૈႣٛǴϪନռண܌ᇨᏤޑମ፦ࢬѨǶӢԜǴ ౦Ч✉़ғނ܈ࢂঁלӆ֎ԏᏊǴૈݯᕍମ፦౧ੱǶҞମ
፦౧ੱҔᛰǴхࡴᚈᕗለᡶᜪǵफ़້ᐟનаϷሟᐟનǴ೭٤ࢂלӆ
֎ԏޑᛰނǴ٠ᙖҗڋᇑମಒझфૈٰᆢମໆ (Goltzman, 2002)Ƕՠ
ࢂ๏ϒ೭٤ᛰނჹܭቚу܈ࡠൺମໆޑቹៜ࣬λǴԃόຬၸ2%
(Rodan and Martin, 2000)Ƕ܌аᇙрзΓᅈཀޑମᏊࢂॶளѐࣴز ޑǴٯӵteriparatideǴѬёаڈᐟཥମޑԋǴ٠অ҅λኺ༾ࢎᄬޑόѳ ᑽǴЪѬࢂᐱբҔܭମ፦౧ੱǶӢࣁཥମғԋЬाфૈࢂٰԾܭ
ମಒझǴ܌аӵ݀ૈቚуମಒझޑϩϯǴ൩ૈቚуମԋբҔ (Cobb and Goldsmith, 1995; Quinn et al., 1998)ǶӧҁጇࣴزǴךॺวٿ ᅿମಒझਲ਼ύǴ౦Ч✉़ғނǴჹܭځϩϯࡰޑALPǵBMP-2ǵ
54
OCаϷOPNԖั༾ޑቹៜǶ܌аҗа่݀ёаޕၰǴ౦Ч✉़ғނ Ԗלӆ֎ԏޑբҔǴ٠Ъૈั༾ቚуମԋޑࢲ܄Ƕ
ಃϤകġ ่ፕ
ҁጇࣴزǴךॺӧମᡎ୷፦༸ಒझϷѮᏘಒझύǴܴ౦Ч✉़
ғނૈڋᇑମಒझԋǶԶ౦Ч✉़ғނΨڋΑRANKL܌ᇨᏤ ޑERKǵp38ǵJNKϷNF-țBࢲ܄ǶӢԜǴ౦Ч✉़ғނ܈ჹܭ෧Ͽ ᇑମಒझԋϷځࢲ܄ǴԖঁၨӳޑቹៜǴ٠ӧମ፦౧ੱݯᕍύǴૈ
ߦମᓐ଼நǶ!
56
ಃΎകġ ୖԵЎ
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ླྀᄪහ ጓǴମ፦౧ੱǴӝკਜрހޗǴѠчǴ1997Ƕ
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Fig. 7 Ipriflavone analogues didn’t affect cell viability.
Bone marrow stromal cells or RAW264.7 cells were treated with ipriflavone analogues (5 ȝM) for 2 days and cell viability was evaluated by the MTT assay. Note that ipriflavone analogues did not affect the cell viability of bone marrow stromal cells and RAW264.7 cells (n=3).
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Osteoclast precursors were isolated from long bones of adult male SD rats and plated on a 24-well plate at 1×106 cells/well. Cultured in the presence of M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 7 days. Following TRAP staining, the cells with more than 3 nuclei were counted (A). Compared with RANKL plus M-CSF, ipriflavone analogues treatment markedly inhibited the differentiation of osteoclast.
The quantitative data are shown in (B)(n=3). RAW264.7 cells were seeded at 2×104 and incubated for 5 days with M-CSF (20 ng/ml) and RANKL (50 ng/ml) without or with ipriflavone analogues. Treatment with ipriflavone analogues inhibited osteoclastogenesis (n=3)(C). RAW264.7 cells were seeded at 2×104 and incubated for 5 days with M-CSF (20 ng/ml) and RANKL (50 ng/ml) without or with ipriflavone analogues. Treatment with ipriflavone analogues inhibited Įvȕ3 expression (n=3)(D). Data are presented as mean±S.E. (n=3). * p<0.05 as compared with control. # p<0.05 as compared with RANKL.
activation.
RAW264.7 cells were pretreated with JFL-142 (0.3 ȝM) and JFL-144 (0.3 ȝM) for 30 min, and followed treatment with RANKL (50 ng/ml) for 60 min, than the p38ˣJNK and ERK phosphorylation was determined by immunoblotting using phospho-p38ˣJNK and ERK specific antibody. Note that ipriflavone analogues inhibits the RANKL-induced p38ˣJNK and ERK phosphorylation .ġ
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RAW264.7 cells were pretreated with JFL-142 (0.3 ȝM) and JFL-144 (0.3 ȝM) for 30 min, and followed treatment with RANKL (50 ng/ml) for 60 min, than the IKKĮ/ȕ, IțBĮ phosphorylation and p65 phosphorylation at Ser536 was determined by immunoblotting using phospho-IKKĮ/ȕ, phospho-IțBĮ and phospho-p65 specific antibody. Note that ipriflavone analogues inhibits the RANKL-induced IKKĮ/ȕ phosphorylation, IțBĮ phosphorylation and p65 phosphorylation (A). RAW264.7 cells were pretreated with JFL-142 (0.3 ȝM) and JFL-144 (0.3 ȝM) for 30 min, before incubation with RANKL (50 ng/ml) for 2 h. The NF-țB binding activity were examined by EMSA (B).
RAW264.7 cells were transfected with țB-luciferase expression vector and then pretreated with ipriflavone analogues for 30 min, before incubation with RANKL (50 ng/ml) for 24 h. Luciferase activity were determined. Data are presented as mean±S.E. (n=3). * p<0.05 as compared with control. # p<0.05 as compared with RANKL.
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MG-63 cells and 3T3 cells were treated with ipriflavone analogues (5 ȝM) for 2 days and cell viability was evaluated by the MTT assay. Note that ipriflavone analogues did not affect the cell viability of MG-63 cells and 3T3 cells. Data are presented as mean±S.E. (n=3).
MG-63 cells and 3T3 cells were treated with ipriflavone analogues for 24 h.
ALP mRNA expression was assessed by qPCR (A炵B). Data are presented as mean±S.E. (n=3). * p<0.05 as compared with control.
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MG-63 cells and 3T3 cells were treated with ipriflavone analogues for 24 h.
BMP-2 mRNA expression was assessed by qPCR (A炵B). Data are presented as mean±S.E. (n=3). * p<0.05 as compared with control.
MG-63 cells and 3T3 cells were treated with ipriflavone analogues for 24 h. OC mRNA expression was assessed by qPCR (A炵B). Data are presented as mean±S.E.
(n=3). * p<0.05 as compared with control.
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MG-63 cells and 3T3 cells were treated with ipriflavone analogues for 24 h.
OPN mRNA expression was assessed by qPCR (A炵B). Data are presented as mean±S.E. (n=3). * p<0.05 as compared with control.
Female BALB/c mice were given a sham operation or ovariectomized. Mice were treated with the ipriflavone or JFL-142 or JFL-144 (100 ȝg/kg) by i.p., than determined the BMC and BMD by DEXA 3 weeks after surgery (A炵B). Data are presented as mean±S.E. (n=8–10 mice/group). * p<0.05 as compared with sham.
# p<0.05 as compared with ovariectomized-group.