Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Expression of Staufen1 and c-myc were determined in human skeletal muscle cells (SkMC), ERMS (RD) and ARMS (RH30) cells. ( a ) Western blot using anti-Staufen1 antibodies and β-actin as a loading control. The predominant Stau1-55 isoform was quantified (n = 4). ( b ) qRT-PCR using primers specific for Staufen1 mRNAs and normalized to total levels of 18 S rRNA (n = 6). ( c ) Western blot using anti-c-myc antibodies and tubulin as a loading control (n = 5). ( d ) qRT-PCR using primers specific for c-myc mRNAs and normalized to total levels of 18 S rRNA (n = 5). All quantifications are represented as a percentage relative to SkMC (n = 5). Data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Analysis of cell proliferation was performed after 72 hours of Control (CTL) or Staufen1-shRNA expression (shStau1) in ERMS and ARMS cells. ( a ) Representative western blot of Staufen1 expression with GAPDH as a loading control in ERMS (RD) and ARMS (RH30) cells. Quantification of n = 4 and n = 3, respectively, is represented as a percentage of the CTL. ( b ) Proliferation was assessed by two-parameter flow cytometry and representative dot plots following 2 h of BrdU incorporation and Propidium Iodide (PI) staining in ERMS (RD) and ARMS (RH30) cells expressing CTL or Staufen1-shRNAs. ( c ) Quantification of BrdU incorporation is represented as a percentage relative to CTL in ERMS (RD) and ARMS (RH30) cells, n = 4 and n = 3, respectively. ( d ) Cell cycle analysis was examined by flow cytometry and the percentage of the cells in G1, S and G2 phases are indicated. Data are Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: shRNA, Expressing, Western Blot, Flow Cytometry, BrdU Incorporation Assay, Staining, Cell Cycle Assay

Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Analysis of apoptosis was performed after 72 hours of Control (CTL) or Staufen1-shRNA expression (shStau1) in ERMS and ARMS cells. ( a ) Representative western blot of Staufen1 expression with GAPDH as a loading control in ERMS (RD) and ARMS (RH30) cells. Quantification of n = 3 is represented as a percentage of the CTL. ( b ) Apoptosis was assessed by two-parameter flow cytometry and representative dot plots of CTL and Staufen1-shRNA expressing ERMS (RD) and ARMS (RH30) cells stained with Annexin V and Propidium Iodide (PI). ( c ) Quantification of Annexin V staining is represented as a percentage relative to CTL (n = 3). Data are Mean ± SEM, *P < 0.05.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: shRNA, Expressing, Western Blot, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Motility and Invasion assays were performed following 48 hours (h) of Control (CTL) or Staufen1-shRNA (shStau1) expression. ( a ) Western blot analysis of Staufen1 expression showing a representative blot of Staufen1 with β-actin as a loading control in ERMS (RD) and ARMS (RH30) cells. ( b ) Cells were seeded into transwell chambers containing membranes coated with or without Matrigel and incubated for 72 h and 48 h for ERMS (RD) and ARMS (RH30) cells, respectively. Representative images of cells that passed through the transwell chamber at 40X magnification, scale bar = 20 μm, are displayed. Average number of cells/field of view is quantified for cell motility (no matrigel) and invasion (matrigel) as a percentage relative to CTL. Migration assays were performed after 48 h of Staufen1 knockdown and ( c ) is a representative western blot and quantification of Staufen1 expression with β-actin as a loading control in ERMS (RD) and ARMS (RH30) cells. ( d ) Cells were seeded into culture insert wells for 24 h, following removal of insert, a 500 μm cell free gap represents 0 h, and consecutive photos were taken at 6 h intervals for a total of 30 h. Representative images at 10X magnification, scale bar = 300 μm. Quantification of the gap is represented as % wound closure normalized to cell confluency. Quantifications are for all data are n = 3. Data are Mean ± SEM, **P < 0.01, ***P < 0.001.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: shRNA, Expressing, Western Blot, Incubation, Migration

Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Flank injections of 6-week-old female SCID-488 mice with ERMS (RD) or ARMS (RH30) cells following 72 hours of Control (CTL) or Staufen1-shRNA (shStau1) expression. To determine Staufen1 knockdown pre-injection, cells were collected for western blot analysis with ( a ) anti-Staufen1 antibodies and GAPDH as a loading control in ERMS (RD) and ARMS (RH30) cells. Quantification is represented as a percentage relative to CTL (n = 1, batch culture). ( b ) Representative endpoint images of ERMS (RD) and ARMS (RH30) at 72 days and 31 days post-injection, respectively. ( c ) Tumour weight from CTL or shStau1 at endpoint for ERMS (RD) (n = 7) and ARMS (RH30) cells (n = 6). ( d ) Tumour volume measurements of ERMS (RD) and ARMS (RH30) tumours over time (n = 7 and n = 6, respectively). Data are Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: shRNA, Expressing, Injection, Western Blot

Journal: Scientific Reports

Article Title: Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

doi: 10.1038/srep42342

Figure Lengend Snippet: Western blot analysis of ERMS (RD) and ARMS (RH30) cells following 48 hours of Control (CTL) or Staufen1-shRNA (shStau1) expression for ( a ) Staufen1 expression ( b ) c-myc expression and ( c ) p 14 ARF. All quantifications are normalized to β-actin and represented as a percentage of the Control (CTL) with n = 3. Polysome profiling of ( d ) ERMS (RD) and ( e ) ARMS cells expressing CTL or shStau1. Polysome profile obtained by continuous absorbance readings at 254 nm (Top) and c-myc mRNA expression measured by qRT-PCR from 10 × 1 mL sucrose gradient fractions (Bottom). Data are Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Alveolar rhabdomyosarcoma RH30 cells (CRL-2061; American Type Culture Collection, VA, USA) and the RH36, RH18 and RH41 cells, which were a gift from Dr. P. Houghton (Department of University of Hematology-Oncology, St Jude Children’s Research Hospital), were cultured in Multicell RPMI 1640 1X with L-Glutamine (Wisent Bioproducts, Quebec, Canada) supplemented with 15% HyClone FBS and 1% HyClone Penicillin-Streptomycin (Thermo Fisher Scientific, Ontario, Canada).

Techniques: Western Blot, shRNA, Expressing, Quantitative RT-PCR

Journal: Orthopaedic Surgery

Article Title: Intermittent Cyclic Mechanical Tension‐induced Down‐regulation of Ectonucleotide Pyrophosphatase Phosphodiesterase 1 Gene Expression is Mainly Dependent on TGF‐β1 in End‐plate Chondrocytes

doi: 10.1111/os.12028

Figure Lengend Snippet: The effect of intermittent cyclic mechanical tension on mRNA expression of ectonucleotide pyrophosphatase phosphodiesterase‐1and end‐plate calcification. (A) Down‐regulation of ENPP‐1 expression in the ICMT‐10 group as assessed by RT‐PCR. The columns represent the mean ± SE *P < 0.05 vs. NC. (B) Alizarin red staining revealed more mineral deposition in the IMCT than in the NC group (×100). NC, normal control.

Article Snippet: The primary antibody used was anti‐enpp‐1 (Catalog no. 2061, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:1000.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Orthopaedic Surgery

Article Title: Intermittent Cyclic Mechanical Tension‐induced Down‐regulation of Ectonucleotide Pyrophosphatase Phosphodiesterase 1 Gene Expression is Mainly Dependent on TGF‐β1 in End‐plate Chondrocytes

doi: 10.1111/os.12028

Figure Lengend Snippet: Expression of ectonucleotide pyrophosphatase phosphodiesterase‐1and transforming growth factor beta 1 with intermittent cyclic mechanical tension‐3. (A) Up‐regulation of ENPP‐1 expression with ICMT‐3 as assessed by real‐time RT‐PCR. (B) Up‐regulation of TGF‐β1 expression in the ICMT‐3 group as assessed by RT‐PCR. The columns represent the mean ± SE. *, P < 0.05, **, P < 0.01 vs. NC. NC, normal control.

Article Snippet: The primary antibody used was anti‐enpp‐1 (Catalog no. 2061, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:1000.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Orthopaedic Surgery

Article Title: Intermittent Cyclic Mechanical Tension‐induced Down‐regulation of Ectonucleotide Pyrophosphatase Phosphodiesterase 1 Gene Expression is Mainly Dependent on TGF‐β1 in End‐plate Chondrocytes

doi: 10.1111/os.12028

Figure Lengend Snippet: Contribution of transforming growth factor beta 1 to regulation of expression of ectonucleotide pyrophosphatase phosphodiesterase‐1with cyclic mechanical strain. (A, B) Total mRNA and proteins were extracted from rat end‐plate exposed to (5, 10) ng/mL of TGF‐β1 for 24 h after ICMT‐3. (C, D) Effect of siRNA on TGF‐β1 induced expression of ENPP‐1. Total mRNA and proteins were extracted from rat end‐plate stimulated with ICMT‐3, in the presence of siRNA on TGF‐β1 for 24 or 48 h. The columns represent the mean ± SE. *, P < 0.05, **, P < 0..01 vs. NC. NC, normal control.

Article Snippet: The primary antibody used was anti‐enpp‐1 (Catalog no. 2061, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:1000.

Techniques: Expressing