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, Control, 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: Control, 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: Control, 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: Control, shRNA, Expressing, Western Blot, Incubation, Migration, Knockdown

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: Control, shRNA, Expressing, Knockdown, 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, Control, shRNA, Expressing, Quantitative RT-PCR

Journal: Carcinogenesis

Article Title: Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner.

doi: 10.1093/carcin/bgp138

Figure Lengend Snippet: Fig. 1. TRPV1 mRNA and protein expression in UC cell lines. (A) TRPV1 mRNA levels from RT4, TCCSUP, J82 and EJ UC cell lines were evaluated by quantitative RT–PCR. TRPV1 mRNA levels (mean ± SD) were expressed as relative fold with respect to NHUC used as control. Values were normalized for b-actin expression. Statistical analysis was performed comparing RT4, TCCSUP, J82 and EJ cell lines with control; P , 0.01. (B) Lysates from NHUC or the indicated UC cell lines were separated on 7.5% SDS–PAGE and probed with a goat anti-human TRPV1 Ab or anti-human a-tubulin mAb. Arrows indicate the bands corresponding to TRPV1 and a-tubulin proteins. Sizes are shown in kilodaltons (kDa). Data are representative of three separate experiments. (C) TRPV1 expression in UC cell lines was evaluated by immunofluorescence and FACS analysis using a goat anti-human TRPV1 Ab. FITC-conjugated RAG was used as secondary Ab. White area indicates secondary Ab alone used as negative control. (D) Immunocytochemical TRPV1 localization in UC cell lines was evaluated by confocal microscopy. Goat anti-human TRPV1 Ab and FITC-conjugated RAG were used as primary and secondary Abs, respectively; bar 5 10 lm.

Article Snippet: Human well-differentiated low-grade papillary RT4 and poorly differentiated, high-grade and muscle invasive UC J82, EJ and TCCSUP cell lines were from American Type Culture Collection (Rockville, MD) and were maintained in RPMI-1640 medium (Flow Laboratories, Irvine, UK) supplemented with 10% heat-inactivated fetal calf serum (Euroclone Ltd, Devon, UK), N-2-hydroxyethylpiperazine N#-2-ethanesulfonic acid, 2mM L-glutamine, 100 IU/ml of penicillin and 100 lg/ml of streptomycin at 37 C, 5% CO2 and 95% of humidity.

Techniques: Expressing, Quantitative RT-PCR, Control, SDS Page, Negative Control, Confocal Microscopy

Journal: Carcinogenesis

Article Title: Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner.

doi: 10.1093/carcin/bgp138

Figure Lengend Snippet: Fig. 2. CPS inhibits cell growth and induces G0–G1 cell growth arrest and apoptosis in RT4 cells. (A) Cell growth was evaluated by MTTassay in RT4, TCCSUP, J82 and EJ cell lines, untreated or treated for 24 h with different doses of CPS (10, 50 and 100 lM). Data are representative of three different experiments. Statistical analysis was performed comparing treated with untreated cells; P , 0.01. (B) Cell growth was evaluated by MTT assay in RT4 cells treated for 24 h with 100 lM CPS alone or in combination with CPZ (10 lM) or with SB366791 (0.1 lM). Control sample indicates dimethyl sulfoxide vehicle-treated cells. Statistical analysis was performed comparing CPS-, CPZ- or SB366791-treated RT4 cells with control and CPS plus CPZ- or CPS plus SB366791- with CPS- treated RT4 cells; P , 0.01. Data are representative of three different experiments. (C) Cell cycle analysis of RT4 cells treated with 100 lM CPS alone or in combination with CPZ (10 lM) or with SB366791 (0.1 lM) was performed by PI staining. Cell percentage relative to different cycle phases is indicated. Data are representative of three different experiments. (D) The percentage of AnnexinVþ RT4 cells treated at different times with 100 lM CPS alone or in combination with CPZ (10 lM) or with SB366791 (0.1 lM), was evaluated by immunofluorescence and FACS analysis. Control sample indicates dimethyl sulfoxide vehicle-treated cells. Data are representative of three different experiments.

Article Snippet: Human well-differentiated low-grade papillary RT4 and poorly differentiated, high-grade and muscle invasive UC J82, EJ and TCCSUP cell lines were from American Type Culture Collection (Rockville, MD) and were maintained in RPMI-1640 medium (Flow Laboratories, Irvine, UK) supplemented with 10% heat-inactivated fetal calf serum (Euroclone Ltd, Devon, UK), N-2-hydroxyethylpiperazine N#-2-ethanesulfonic acid, 2mM L-glutamine, 100 IU/ml of penicillin and 100 lg/ml of streptomycin at 37 C, 5% CO2 and 95% of humidity.

Techniques: MTT Assay, Control, Cell Cycle Assay, Staining

Journal: FEBS Open Bio

Article Title: CORO1C expression is associated with poor survival rates in gastric cancer and promotes metastasis in vitro

doi: 10.1002/2211-5463.12639

Figure Lengend Snippet: CORO1C promoted cell proliferation of human gastric cancer cells. BGC‐823 and AGS cells were transfected with si CORO1C‐1 , si CORO1C‐2 or si NC . (A,B) Protein levels of CORO1C were detected by western blot. β‐Actin was used as control. (C,D) Cell total number assay was performed with an original cell number of 1 × 10 5 . Cells were counted every day for 5 days. (E,F) MTT assay was performed to evaluate cell viability (BGC‐823‐si NC 100.00%; BGC‐823‐si CORO1C‐1 39.18%, BGC‐823‐si CORO1C‐2 39.30%, both P < 0.01 vs si NC ; AGS‐si NC 100.00%; AGS‐si CORO1C‐1 35.31%, AGS‐si CORO1C‐2 32.67%, both P < 0.01 vs si NC ). (G,H) Cell colony formation assay was performed with an original cell number of 1000, and cell colony numbers were calculated after 10 days (BGC‐823‐si NC 100.00%; BGC‐823‐si CORO1C‐1 39.84%, BGC‐823‐ si CORO1C‐2 36.54%, both P < 0.01 vs si NC ; AGS‐si NC 100.00%; AGS‐si CORO1C‐1 28.68%, AGS‐si CORO1C‐2 27.67%, both P < 0.01 vs si NC ). Scale bar, 200 μm. All data are shown as the mean ± standard deviation and were evaluated using unpaired two‐tailed t test. Every test was repeated at least three times and the results represented the average ( n ≥ 3). ** P < 0.01.

Article Snippet: Human gastric cancer cell lines BGC‐823 and AGS [obtained from ATCC (the American Type Culture Collection) Rockville, MD, USA] were used in this study.

Techniques: Transfection, Western Blot, Control, MTT Assay, Colony Assay, Standard Deviation, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CORO1C expression is associated with poor survival rates in gastric cancer and promotes metastasis in vitro

doi: 10.1002/2211-5463.12639

Figure Lengend Snippet: CORO1C inhibited cellular apoptosis and facilitated cell mitosis of human gastric cancer cells. BGC‐823 and AGS cells were transfected with si CORO1C‐1 , si CORO1C‐2 or si NC . (A) Cellular apoptosis analysis was performed by flow cytometry. (B,C) Cell cycle analysis was performed by flow cytometry. All data are shown as the mean ± standard deviation and were evaluated using unpaired two‐tailed t test. Every test was repeated at least three times and the results represented the average ( n ≥ 3). ** P < 0.01.

Article Snippet: Human gastric cancer cell lines BGC‐823 and AGS [obtained from ATCC (the American Type Culture Collection) Rockville, MD, USA] were used in this study.

Techniques: Transfection, Flow Cytometry, Cell Cycle Assay, Standard Deviation, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CORO1C expression is associated with poor survival rates in gastric cancer and promotes metastasis in vitro

doi: 10.1002/2211-5463.12639

Figure Lengend Snippet: CORO1C promoted cell migration and invasion of human gastric cancer cells. BGC‐823 and AGS cells were transfected with si CORO1C‐1 , si CORO1C‐2 or si NC . (A,B) Cell migration assay was performed; 1 × 10 5 cells were seeded in transwells and cell migration was detected after 24 h in both BGC‐823 and AGS cells (BGC‐823‐si NC 100.00%; BGC‐823‐si CORO1C‐1 44.26%, BGC‐823‐si CORO1C‐2 40.24%, both P < 0.01 vs si NC ; AGS‐si NC 100.00%; AGS‐si CORO1C‐1 40.60%, AGS‐si CORO1C‐2 34.90%, both P < 0.01 vs si NC ). Scale bar, 200 μm. (C,D) Cell invasion assay was performed; 1 × 10 5 cells were seeded in transwells with Matri‐gel and cell invasion was detected after 48 h in both BGC‐823 and AGS cells (BGC‐823‐si NC 100.00%; BGC‐823‐si CORO1C‐1 33.69%, BGC‐823‐ si CORO1C‐2 28.96%, both P < 0.01 vs si NC ; AGS‐si NC 100.00%; AGS‐si CORO1C‐1 41.63%, AGS‐si CORO1C‐2 34.69%, both P < 0.01 vs si NC ). Scale bar, 200 μm. All data are shown as the mean ± standard deviation and were evaluated using unpaired two‐tailed t test. Every test was repeated at least three times and the results represented the average ( n ≥ 3). ** P < 0.01.

Article Snippet: Human gastric cancer cell lines BGC‐823 and AGS [obtained from ATCC (the American Type Culture Collection) Rockville, MD, USA] were used in this study.

Techniques: Migration, Transfection, Cell Migration Assay, Invasion Assay, Standard Deviation, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CORO1C expression is associated with poor survival rates in gastric cancer and promotes metastasis in vitro

doi: 10.1002/2211-5463.12639

Figure Lengend Snippet: CORO1C promoted the expression of cyclin D1 and vimentin in gastric cancer cells. (A) mRNA levels of EZH2 , BCL‐2 , cyclin D1 , MDM2 , vimentin and C‐myc in BGC‐823 cells after transfected with si CORO1C‐1 , si CORO1C‐2 or si NC were detected using RT‐qPCR. (B,C) BGC‐823 and AGS cells were transfected with si CORO1C‐1 or si NC . Protein levels of CORO1C, cyclin D1 and vimentin were detected using western blot. β‐Actin was detected as control. All data are shown as the mean ± standard deviation and were evaluated using unpaired two‐tailed t test. Every test was repeated at least three times and the results represented the average ( n ≥ 3). ** P < 0.01.

Article Snippet: Human gastric cancer cell lines BGC‐823 and AGS [obtained from ATCC (the American Type Culture Collection) Rockville, MD, USA] were used in this study.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, Standard Deviation, Two Tailed Test