Journal: Nature

Article Title: Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.

doi: 10.1038/nature08792

Figure Lengend Snippet: Figure 2 | Telomere elongation in DKC1 mutant iPS cells. a, Telomere Southern blot of del37L fibroblasts (Fib) and iPS clones 1 and 2 as a function of passage. MM, molecular mass marker. b, Quantitative real-time PCR (qPCR) analysis of telomere length in del37L fibroblasts and iPS clones at indicated passages (p). Error bars represent s.e.m. c, Quantitative fluorescence in situ hybridization (qFISH) for telomere length in del37L cells. a.f.u., arbitrary fluorescence units. Inset, mean relative length values shown 6s.d.

Article Snippet: The DKC1 retroviral vector was created by cloning a full-length DKC1 cDNA (OriGene) into pMIG-W (Addgene) and retrovirus was produced as described31.

Techniques: Mutagenesis, Southern Blot, Clone Assay, Marker, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: Cancers

Article Title: Aberrant Dyskerin Expression Is Related to Proliferation and Poor Survival in Endometrial Cancer

doi: 10.3390/cancers13020273

Figure Lengend Snippet: DKC1 mRNA and dyskerin protein in human endometrium. ( A ) DKC1 mRNA is normalised to geometric means of PPIA and YWHAZ and measured by qPCR in endometrial tissue samples: healthy postmenopausal (PM) ( n = 6) and endometrial cancer (EC) ( n = 22). Mann-Whitney test. ( B ) The amount of dyskerin protein was evaluated by immuno-blotting in healthy PM ( n = 4) and EC ( n = 4), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used to ensure equal loading of protein. Dyskerin protein levels in epithelial cells of tissue samples were analysed by normalising to pancytokeratin (panck). Mann-Whitney test, * p < 0.05. ( C ) Telomerase activity (TA) in healthy endometrial PM ( n = 6) and EC ( n = 32) was measured using a Telomere Repeat Amplification Protocol (TRAP) assay, Mann-Whitney test, ** p < 0.01. AU: arbitrary units ( D ) Representative microphotographs illustrating dyskerin IHC staining at the cellular level in endometrial samples in ( 1 ) normal proliferative phase (PP) endometrium, ( 2 ) healthy PM endometrium, ( 3 ) endometrial hyperplasia with cytological atypia (EHA) and ( 4 ) EC. Positive staining appears brown. Magnification 400×. Scale bar 50 μm. ( E ) Immunostaining quickscores for dyskerin protein in the human endometrium, healthy PP ( n = 16), PM ( n = 30), EHA ( n = 15), EC ( n = 109). Kruskal-Wallis test, * p < 0.05, **** p < 0.0001.

Article Snippet: Transfection of ISK cells was performed twenty-four hours after seeding cells on 6 well plates at a density of 0.5 × 10 6 cells/well by using a mixture of MYC-DDK tagged Dyskerin plasmid (OriGene Technologies, Rockville, MD, USA, 3 μL) with Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK, 9 μL).

Techniques: MANN-WHITNEY, Activity Assay, Amplification, TRAP Assay, Immunohistochemistry, Staining, Immunostaining

Journal: Cancers

Article Title: Aberrant Dyskerin Expression Is Related to Proliferation and Poor Survival in Endometrial Cancer

doi: 10.3390/cancers13020273

Figure Lengend Snippet: Immunostaining of dyskerin in endometrial cancer subtypes ( n = 109). ( A ) Representative microphotographs of dyskerin in human ECs. ( 1 – 3 ) grade 1–3 endometrioid carcinoma, ( 4 ) serous subtype, ( 5 ) Carcinosarcoma and ( 6 ) clear cell carcinoma. Positive staining appears brown. Magnification 400×. Scale bar 50 μm. ( B ) Dyskerin immunoscores in healthy PM ( n = 30) and various EC subtypes including endometrioid (E) ( n = 65), Serous (S) ( n = 12), carcinosarcoma (CS) ( n = 19), clear cell carcinoma ( C ) ( n = 10), mixed cell adenocarcinoma (M) ( n = 2) and dedifferentiated EC (DD) ( n = 1). ** p < 0.01, **** p < 0.0001. Kruskal-Wallis test. ( C ) Dyskerin immunoscores in human endometrial epithelium of healthy PM ( n = 30), LGEC ( n = 53) and HGEC ( n = 56). *** p < 0.001. Kruskal-Wallis test.

Article Snippet: Transfection of ISK cells was performed twenty-four hours after seeding cells on 6 well plates at a density of 0.5 × 10 6 cells/well by using a mixture of MYC-DDK tagged Dyskerin plasmid (OriGene Technologies, Rockville, MD, USA, 3 μL) with Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK, 9 μL).

Techniques: Immunostaining, Staining

Journal: Cancers

Article Title: Aberrant Dyskerin Expression Is Related to Proliferation and Poor Survival in Endometrial Cancer

doi: 10.3390/cancers13020273

Figure Lengend Snippet: Dyskerin immunostaining in endometrial cancers. ( A ) Representative microphotographs illustrating dyskerin immunohistochemical staining in primary endometrial cancer (EC) ( 1 ) and matched metastatic lesion ( 2 ). Positive staining appears in brown. Magnification 400×, Scale bar 50 μm ( B ) Difference in dyskerin immunoscores in primary EC samples versus matched metastatic lesions ( n = 30) each, ** p < 0.01. ( C ) Difference in dyskerin immunoscores between early-stage ECs (FIGO stage I–II) ( n = 63) and advanced stage ECs (FIGO stage III–IV) ( n = 43). Mann-Whitney test, * p < 0.05.

Article Snippet: Transfection of ISK cells was performed twenty-four hours after seeding cells on 6 well plates at a density of 0.5 × 10 6 cells/well by using a mixture of MYC-DDK tagged Dyskerin plasmid (OriGene Technologies, Rockville, MD, USA, 3 μL) with Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK, 9 μL).

Techniques: Immunostaining, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Cancers

Article Title: Aberrant Dyskerin Expression Is Related to Proliferation and Poor Survival in Endometrial Cancer

doi: 10.3390/cancers13020273

Figure Lengend Snippet: Kaplan Meier survival curves for the correlation between dyskerin immunoscores and patient outcome. ( A ) Disease-free survival (DFS), the median DFS time is undefined for low dyskerin and high dyskerin endometrial cancer groups. Hazard ratio (HR) = 1.92, 95% CI of the ratio (0.9200–4.006) ( B ) Cancer-specific survival (CSS), the median CSS time was undefined for low dyskerin and high dyskerin endometrial cancer groups. HR = 1.991, 95% CI of HR (0.9300–4.261) and ( C ) Overall survival (OS) in endometrial cancer samples ( n = 109). Median OS time: Low dyskerin protein 8.00 months, High dyskerin protein 2.00 months. Low dyskerin/high dyskerin median survival Ratio: 0.5217, 95% CI of ratio (0.004444–1.039) HR = 1.841, 95% CI of HR (0.9667–3.506). A quickscore of 6 was chosen as the cut-off point. The p values relevant to the difference between low and high dyskerin protein levels in endometrial cancer groups that is visually represented in Kaplan Meier survival curves from the log-rank test.

Article Snippet: Transfection of ISK cells was performed twenty-four hours after seeding cells on 6 well plates at a density of 0.5 × 10 6 cells/well by using a mixture of MYC-DDK tagged Dyskerin plasmid (OriGene Technologies, Rockville, MD, USA, 3 μL) with Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK, 9 μL).

Techniques:

Journal: Cancers

Article Title: Aberrant Dyskerin Expression Is Related to Proliferation and Poor Survival in Endometrial Cancer

doi: 10.3390/cancers13020273

Figure Lengend Snippet: Transient overexpression of DKC1 in ISK cells. The plasmid and the empty vector (EV) used were tagged with the synthetic DYKDDDDK (DDK) protein to discern the transfected cells by using an anti-DDK antibody. ( A ) Immunoblot showing the level of dyskerin protein in DKC1 and EV transfected and non-transfected (NT) ISK cells. Cells were harvested 6, 24, and 48 h following transfection. Endogenous and exogenous dyskerin bands were present at the molecular weight of 58 and 60 KDa (red and blue arrows, respectively). DDK bands (yellow arrows) were observed at 60 KDa. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) bands were at 37 KDa. ( B ) Flow cytometric histogram showing the level of DDK tag protein in ISK cells. Cells positively stained with anti-DDK tag antibody represent transfected cells. ( C ) Cell proliferation was analysed using flow cytometry. ISK cells were stained with CellTrace Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) and fluorochrome-conjugated DDK Tag Antibody. Transfected cells (blue curve) and non-transfected cells (red curve). Higher proliferation is suggested when the curve was shifted to the left. ( D ) The difference in median fluorescence index (MFI) between transfected (T) and non-transfected ISK cells. ** p < 0.01, Wilcoxon signed-rank test.

Article Snippet: Transfection of ISK cells was performed twenty-four hours after seeding cells on 6 well plates at a density of 0.5 × 10 6 cells/well by using a mixture of MYC-DDK tagged Dyskerin plasmid (OriGene Technologies, Rockville, MD, USA, 3 μL) with Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK, 9 μL).

Techniques: Over Expression, Plasmid Preparation, Transfection, Western Blot, Molecular Weight, Staining, Flow Cytometry, Fluorescence

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Dyskerin KD stimulates VEGF-IRES–mediated translation. Transient transfection of DKC1-specific siRNA strongly reduced DKC1 mRNA and protein level in MCF7 and MDA-MB231 cells ( A , left and B , left, respectively). [3H]-leucine incorporation indicates that the total protein synthesis is not compromised (A and B, center) after DKC1 KD. IRES-mediated translation was assessed by measuring the FLuc and RLuc activity in MCF-7 and MDA-MB231 (A, right, and B, right, respectively) cells 8 h after the transfection with the bicistronic mRNA transcribed from pRL-VEGF-IRES. siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from at least three independent experiments. P < 0.05 are considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Transfection, Activity Assay, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Dyskerin KD drives VEGF mRNA translation in breast cancer cells. Total (left) and polysome-associated (right) VEGF mRNA levels assessed by real-time PCR after DKC1 KD in MCF7 ( A ) and MDA-MB231 cells ( C ). Representative polysomal profiles are shown. VEGF protein levels in supernatant and in whole cell extracts are also reported for MCF-7 ( B ) and MDA-MB231 cells ( D ). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: The increased VEGF secretion in DKC1 KD cells is due to the up-regulation of VEGF mRNA IRES-mediated translation. ( A ) Histograms show the level of VEGF protein detected using an ELISA array. Experiments were performed in MCF-7 control (left) and DKC1 KD (right) cells treated with 15 nM PTC299 for 72 h. VEGF secretion of PTC299 treated cells is normalized on the correspondent untreated cells; for the effect of DKC1 KD on VEGF secretion on MCF7 cells see A ( B ) VEGF-IRES–mediated translation measured in MCF7 control and DKC1 KD cells after 72 h of 100 nM PTC299 treatment. siRNA transfection was performed 96 h before cell harvesting. ( C ) Representative image (left) and a summarizing graph (right) from clonogenic assays performed with control (empty vector) and shDKC1 MCF7 cells. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, Cell Harvesting, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: DKC1 KD increases VEGF-IRES recruitment to 48S preinitiation complex. ( A ) Left: representative profile at 260 nM O.D. obtained from MCF7 cytoplasmic extracts: fractions 18–21 were considered to correspond to the small ribosomal subunits. Right: Representative profile of a sucrose density gradient reporting the radioactive intensity per fraction in from DKC1 KD (circles) and control (SCR—triangles) cells extracts, respectively. Peaks of radioactivity coinciding with the identified fractions containing the 48S complexes was generated when MCF7 cytoplasmic extracts were incubated with a [ 32 P]VEGF IRES mRNA probe. ( B ) Histogram represent mean and SD of the radioactivity measured in the identified peaks for DKC1 KD and control (SCR) cells extracts. siRNA transfection was performed 96 h before cell harvesting. P < 0.05 is considered significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Control, Radioactivity, Generated, Incubation, Transfection, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Reduction of dyskerin levels differentially affects IRES-mediated translation of viral and cellular IRESs. ( A ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells, 8 h after transfection with a bicistronic mRNA transcribed from viral pR-CrPV-IRES-F (left), pR-HCV-IRES-F (center) and pF-EMCV-IRES-R (right). ( B ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells 8 h after transfection with a bicistronic mRNA transcribed from cellular pR-HSP70-IRES-F (left), pR-c-MYC-IRES-F (center) and pR-p53IRES-F (right). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Activity Assay, Transfection, Cell Harvesting