Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: Osteoblast adhesion and survival are markedly reduced and Rictor expression is downregulated with aging in vitro and in vivo . ( a ) Representative immunohistochemical (IHC) staining for osteocalcin (OCN) from 3-month-old and 16-month-old mice ( n =6). Arrow, osteoblast; BS, bone surface; scale bar, 50 μ m; * P <0.01 versus 3M. ( b ) SEM image of femora trabecular bones from 3- and 16-month-old mice. Upper panel showed less and thinner trabecula bone in 16-month-old mice. Black arrow, osteoblasts or osteocytes, up panel scale bar, 500 μ m; lower panel scale bar, 50 μ m. ( c ) Photomicrographs of fluorescent calcein staining of 3- and 16-month-old mice and quantification of mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR) and MAR/osteoblast. Arrow: bone formation surface; scale bar, 20 μ m ( n =3 * P <0.01 versus 3M). ( d ) Cell adhesion analyses of calvarial osteoblast cultures from 3- and 16-month-old mice with (lower panel) or without (upper panel) matrigel in the plate ( n =3 * P <0.01 versus 3M). ( e ) Representative photomicrographs of bone nodule formation in MSC cultures from 3- and 16-month-old mice. Scale bar, 400 μ m. ( f ) Western blot analysis of cleaved-poly ADP-ribose polymerase (PARP) in lysates of trabecular bone dissected from femora of 3- and 16-month-old mice. ( g ) Western blot analysis of Rictor and P-Akt(S473) expression in lysates of trabecular bone from 3- and 16-month-old mice. ( h ) Western blot analysis of mTOR, Rictor, Raptor and P-Akt(S473) expression in lysates of lung, spleen and kidney from 3- and 16-month-old mice. ( i ) Western blot analysis of Rictor, mTOR, Raptor, osteocalcin (OCN) and P-Akt(S473) expression in primary calvarial osteoblasts from 3- and 16-month-old mice

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, In Vitro, In Vivo, Immunohistochemical staining, Immunohistochemistry, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: Rictor is a target of miR-218 in osteoblasts. ( a ) Real-time PCR analysis of 13 miRNAs expression in trabecular bone from 3- and 16-month-old mice ( n =6). * P <0.01 versus 3M. ( b ) Western blot analysis of Rictor, p-Paxillin(Y118) and P-Akt(S473) expression in osteoblasts transfected with miR-218 mimics and miR-218 inhibitors. ( c ) The putative miR-218 binding site in the Rictor 3′ UTR. ( d ) Luciferase activity in MC3T3-E1 cells co-transfected with miR-218 mimics or negative control (NC) and the indicated 3′ UTR-driven reporter constructs ( n =3). * P <0.01 versus NC; ** P <0.05 versus NC

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Binding Assay, Luciferase, Activity Assay, Negative Control, Construct

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: miR-218 represses osteoblast adhesion and survival by targeting Rictor. ( a ) Adhesion analysis of MC3T3-E1 cells transfected with miR-218 mimics, miR-218 inhibitors and NCs ( n =3). * P <0.01, ** P <0.05. ( b ) AnnexinV analysis of apoptosis in MC3T3-E1 cells transfected with miR-218 mimics or miR-218 inhibitor and serum-starved for 48 h ( n =3). * P <0.01, ** P <0.05. ( c ) Western blot analysis of Rictor, p-Paxillin(Y118), Runx2 and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 mimics. ( d ) Western blot analysis of Rictor, p-Paxillin(Y118), Runx2 and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 inhibitor or NCs. ( e ) Western blot analysis of Rictor, p-Paxillin(Y118) and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vectors. ( f ) Adhesion analysis of MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vector ( n =3). * P <0.01, ** P <0.05. ( g ) AnnexinV analysis of apoptosis in MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vector ( n =3); * P <0.01, ** P <0.05

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: ROS stimulates miR-218 to downregulate Rictor in aged mice osteoblast. ( a ) ROS levels in trabecular bones of 3- and 16-month-old mice ( n =6); * P <0.01 compared with 3-month-old mice. ( b ) Effect of H 2 O 2 (1 μ M) on miR-218 expression in MC3T3-E1 cells ( n =3). * P <0.01 versus controls. ( c ) Western blot analysis of Rictor, mTOR and Raptor expression in MC3T3-E1 cells treated with H 2 O 2 (5, 10 μ M) for 48 h. ( d ) Effect of H 2 O 2 (1 μ M for 24 h) on MC3T3-E1 cell adhesion ( n =3). * P <0.01 versus controls. ( e ) Effect of H 2 O 2 (10 μ M for 36 h) and on MC3T3-E1 cell apoptosis( n =3). * P <0.01 versus controls

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: ROS scavenger reduces miR-218 and Rictor expression and aged-related bone loss in mice. ( a ) Western blot analysis of Rictor, Raptor and P-Akt(S473) expression in trabecular bone of 3-, 9- and 16-month-old mice. ( b ) Trabecular bone ROS levels in 9-month-old mice treated with N-acetyl- l -cysteine (NAC; 2 mg/ml) or vehicle for 7 months ( n =10). * P <0.01 versus controls. ( c ) Representative micro-CT scans of vertebrae from mice described in ( b ). ( d – g ) Trabecular bone BV/TV, BMD, Tb.Sp and Tb.N in mice described in ( c ) ( n =10); * P <0.01 versus controls. ( h , i ) Oestocalcin IHC ( h ) or TRAP ( i ) staining of distal femora from mice described in ( b ). Scale bar, 50 μ m; * P <0.01 versus controls. ( j ) Real-time PCR analysis of miR-218 expression in trabecular bones of mice described in ( b )( n =10). * P <0.01 versus controls. ( k ) Westem bolt analysis of c-PARP expression in trabecular bones of mice described in (b). ( l ) Western blot analysis of Rictor, Raptor and P-Akt(S473) expression in trabecular bones of mice described in ( b ). ( m ) A schematic model depicting that the upregulation of miR-218 and loss of Rictor with aging induces the pathogenesis of age-related bone loss

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Micro-CT, Staining, Real-time Polymerase Chain Reaction