Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YAP transcriptionally regulates COX-2 expression and GCCSysm-4 (G-4), a dual YAP/COX-2 inhibitor, overcomes drug resistance in colorectal cancer

doi: 10.1186/s13046-017-0612-3

Figure Lengend Snippet: G-4 (10 μM) disturbed YAP-TEAD interaction. a G-4 treatment disturbed the YAP-TEAD1 interaction in the nucleus of HCT15/Tax cells. The YAP-TEAD1 interaction was probed in cells 4 h after G-4 treatment and in untreated cells using co-IP. b ChIP analysis of YAP interaction with the Cyr 61 and COX-2 promoter in HCT15/Tax cells. YAP was examined in cells 4 h after G-4 treatment and in untreated cells. ** P < 0.01 compared with vehicle group. c Cyr 61 and CTGF luciferase reporter activity was measured after treatment with G-4 for 48 h. The fold changes in luciferase activity were calculated by normalizing untreated cells with G-4-treated cells. Data are representative of at least three independent experiments. Error bars represent SD. ** P < 0.01 compared with control cells

Article Snippet: DNA plasmids that encode wild type human YAP (hYAP, CMV2-YAP) and TEAD1 vector (pRK5-Myc-TEAD 1) and YAP, COX-2, LATS1 shRNA were obtained from Addgene (USA).

Techniques: Co-Immunoprecipitation Assay, Luciferase, Activity Assay, Control

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

Journal: Cell reports

Article Title: The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

doi: 10.1016/j.celrep.2020.108230

Figure Lengend Snippet: Figure 4. Active mTORC1 Promotes Survival in Response to Mitotic Poisons (A) HeLa cells were transfected in triplicate with either pRK5-myc raptor WT or 8A mutant and synchronized using thymidine/nocodazole protocol, then labeled with [S35] methionine/cysteine, and the specific activity of incorporation into equal amounts of protein was determined by trichloroacetic acid (TCA) precipitation and scintillation counting. Data are expressed as means ± SEMs. Comparison was calculated using unpaired two-tailed Student’s t test (*p < 0.01). (B) Immunoblot analysis of HeLa cells that were transfected and synchronized as in (A). Nocodazole-arrested cells were collected and released into fresh media. Degradation of cyclin B1 and phosphorylation of cdc27 are used as markers for exit from mitosis. (C) The length of one whole cell cycle (between two mitoses) was measured for 50 cells (transfected with EGFP raptor WT or 8A) using time-lapse microscopy. Comparison was calculated using unpaired two-tailed Student’s t test (ns, p > 0.05). (D) HeLa cells were transfected with either EGFP-empty vector or Raptor WT or 8A mutant. Twenty-four hours following transfection, cells were synchronized by RO3306. After 20 h, cells were washed twice with PBS and released into fresh media containing either 100 nM Taxol (top panel) or 100 nM Taxol + 200 nM rapamycin, and time-lapse imaging was started. The length of time spent by 100 cells from mitotic entry until death was plotted. Mean death time is shown as a point estimate ± SEM and as a bar plot (right panel) for each treatment. Comparisons were calculated using one-way ANOVA with Bonferroni’s multiple com- parison tests (*p < 0.01; ns, p > 0.05).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Plasmid: pcDNA3-Flag mTOR wt (Vilella-Bach et al., 1999) Addgene Plasmid # #26603 Plasmid: myc-mTOR (Sarbassov et al., 2004) Addgene Plasmid # #1861 Plasmid: pRK5 Flag PRAS40 (Sancak et al., 2007) Addgene Plasmid # #14950 Plasmid: pRK5-myc-PRAS40 (Vander Haar et al., 2007) Addgene Plasmid # #15476 Plasmid: pRK5- myc-empty vector Dr.

Techniques: Transfection, Mutagenesis, Labeling, Activity Assay, TCA Precipitation, Comparison, Two Tailed Test, Western Blot, Phospho-proteomics, Time-lapse Microscopy, Plasmid Preparation, Imaging