ho 1 sirna sequences  (Shanghai Generay Biotech)

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: ( A ) HUVEC were transfected with control siRNA (CTL) or HO-1-specific siRNA prior to culture in the presence or absence of VEGF (25 ng/ml) for 48 h. Proliferation was quantified by flow-cytometric analysis of PCNA staining. ( B ) HUVEC were left uninfected or infected with the Ad0 control adenovirus or AdHO-1 (multiplicity of infection (MOI) 100 ifu/cell). EC were then cultured in the absence or presence of VEGF for 48 h and proliferation quantified by BrdU ELISA. ( C ) HUVEC were treated with vehicle alone (dotted line) or transfected with control siRNA (CTL) or HO-1 specific siRNA (seq2 or 4 pooled sequences (pool)). Cells were cultured in M199 medium/10% FCS (white bars) or serum-starved in M199/0.1% BSA in the absence or presence of VEGF (25 ng/ml) for 48 h (dark bars). Cell number was assessed by MTS-assay. ( D ) HUVEC were transfected with control (CTL) or pooled HO-1 siRNA oligos (HO-1) as above prior to culture with or without VEGF and serum starvation. Sub-G1 apoptotic EC were identified by propidium iodide staining and quantified by flow-cytometry, with data expressed as a percentage of the apoptosis seen in control siRNA-treated serum-starved cells. ( E,F ) HUVEC were transfected with control or pooled HO-1 siRNA (HO-1) and cultured in the absence or presence of VEGF for 48 h, with (E) Bcl-2 protein analysed by immunoblotting and quantified by densitometry, and ( F ) A1 mRNA quantified by qRT-PCR. Data are presented as mean ± SEM (n ≥ 4 experiments), * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Staining, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, MTS Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: HUVEC were transfected with 3′-AlexaFluor488-labelled control siRNA (CTLAF 488 ) or HO-1-specific siRNA (seq2AF 488 ) and ( A ) Confluent EC monolayers were scratched and migration (Euclidean distance) was assessed by live cell imaging in the absence or presence of VEGF (25 ng/ml) for 16 h. ( B,C ) Illustrate directional migration, with images representative of n = 3 experiments, ( B ) Ctl siRNA and ( C ) HO-1 siRNA, with histograms plotting migration of individually tracked cells. Bar = 50 μm. ( D ) HUVEC were infected with Ad0 or AdHO-1 (MOI 100) and cultured for 16 h. Confluent cell monolayers were scratched and migration assessed in the absence or presence of VEGF as above for 16 h. ( E,F ) HUVEC were transfected with CTLAF 488 ( E ), or ( F ) HO-1 siRNA (seq2AF 488 ) and re-seeded onto collagen IV-coated Ibidi chemotaxis μ-slides. The upper reservoir contained EBM-2 medium with 25 ng/ml VEGF and the lower reservoir plain EBM-2. Tracking of fluorescent transfected cells was captured by time-lapse microscopy every 15 mins for 16 h. Migration paths along the VEGF gradient are shown in black and away in red. Plots are representative of 3 experiments and ( G ) shows quantitative data presented as mean ± SEM (n = 3). Migration was quantified using ImageJ software (Manual Tracking, Chemotaxis and Migration tool). Data are presented as mean ± SD (n ≥ 3 experiments), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Migration, Live Cell Imaging, Infection, Cell Culture, Chemotaxis Assay, Time-lapse Microscopy, Software

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: ( A ) HUVEC were transfected with control siRNA (CTL), HO-1 siRNA (seq2) or pooled HO-1 siRNAs (pool). Cells were seeded onto growth factor-reduced Matrigel in the presence or absence of VEGF for 16 h. Images from five fields per view were captured, and ( B ) tube length was quantified using ImageJ software (NeuronJ). ( C ) Murine EC from Hmox1 −/− mice or Hmox1 +/+ littermate controls were seeded onto growth factor-reduced Matrigel in the presence or absence (UT) of VEGF for 16 h. Images from five fields per view were captured and ( D ) tube length quantified using ImageJ. Pictures are representative of 3 experiments (Bars = 50 μm). Data are presented as mean ± SEM (n = 3 experiments), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Software

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: ( A ) Control siRNA (siCTL)-transfected or pooled siHO-1-transfected HUVEC were left untreated or treated with 25 ng/ml VEGF for 48 h prior to propidium iodide staining and flow-cytometric analysis of DNA distribution. Data is presented as mean ± SEM (n = 3 experiments), * p < 0.05 vs siCTL, δ p < 0.05 vs siCTL + VEGF. ( B ) HUVEC were exposed to geneFECTOR alone (GF) or transfected with control siRNA (CTL) or pooled HO-1 siRNA (HO-1). Cells were cultured in the presence or absence of VEGF for 24 h prior to mRNA quantification by qRT-PCR of ( B ) cyclin A1, ( C ) cyclin E1, ( D ) cyclin-dependent kinase 2 (cdk2) or ( E ) p27. Data are presented as mean ± SEM (n = 3 experiments), * p < 0.05, ** p < 0.01. δδ p < 0.01 vs untreated siCTL-transfected cells.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Staining, Cell Culture, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: HUVEC were transfected with CTLAF 488 or HO-1 siRNA (seq2AF 488 ). Monolayers were scratched and treated with vehicle alone or VEGF (25 ng/ml) for 16 h ( A ) Migration was quantified by live cell imaging using ImageJ. EC were then harvested, lysed and immunoblotted for: ( B ) cyclin A1 and ( C ) retinoblastoma protein (pRb) and phospho-pRb pT821 . The histograms show corresponding densitometry data corrected for the GAPDH loading control and with phospho-pRb expressed relative to total pRb. Data are presented as mean ± SEM (n = 3 experiments), * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Migration, Live Cell Imaging

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: Female C57BL/6 mice (10 weeks old, n = 3 per treatment) were injected with Matrigel preparations containing: ( A ) vehicle control, ( B ) VEGF (40 ng/ml) with 2 μM control siRNA (CTL), ( C,D ) VEGF with murine HO-1 siRNA (HO-1), ( E,F ) VEGF with murine CycA1 siRNA (CycA1). ( A,B,C,E ) 10x magnification, bar = 50 μm ( D,F ) 20x magnification, bars = 100 μm. In ( G–J ) Matrigel plugs contained ( G ) Ad0 control, ( H ) AdHO-1, ( I ) AdHO-1 + Ctl siRNA, ( J ) AdHO-1 + CycA1 siRNA. Representative images (10x magnification, bars = 50 μm). ( K ) Area occupied by neovessels quantified using ImageJ software. Data are presented as mean ± SEM (n = 3 experiments). * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Injection, Software

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: ( A,B ) HUVEC were treated with geneFECTOR (GF) alone or transfected with control siRNA (CTL) or pooled HO-1 siRNAs (HO-1) prior to treatment with VEGF (25 ng/ml) or vehicle for 16 h and: ( A ) HO-1 and vimentin were quantified by immunoblotting, and in ( B ) Vimentin and HO-1 were assessed by Phos-tag SDS-PAGE and immunoblotting. Changes in expression were quantified by densitometry corrected for GAPDH. ( C ) HUVEC were either treated with GF alone or transfected with CTL or HO-1 siRNAs (seq2 or pool) for 24 h. Cells were pre-loaded with calpain substrate tBoc-LM-CMAC prior to treatment with VEGF or vehicle. In ( D ) HUVEC were pre-loaded with the calpain substrate prior to treatment with hemin (0.2 or 2 μM) for 2 h. Calpain activity was assessed by flow-cytometry. Data are presented as the mean fluorescence intensity (MFI) ± SEM (n = 3 experiments)* p < 0.05,** p < 0.01, *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Western Blot, SDS Page, Expressing, Activity Assay, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: HUVEC, transfected with ( A,B ) CTLAF 488 or ( C,D ) HO-1 siRNA seq2AF 488 , were seeded onto gelatin-coated coverslips in the absence (UT) or the presence of 25 ng/ml VEGF for 16 h, prior to fixation and vimentin staining. Nuclei were stained with Draq5. Immunofluorescence was analyzed by confocal microscopy. Representative images are shown, 63x magnification, bar = 50 μm. ( E ) Cell area was quantified using ImageJ (n = 15). ( F ). HUVEC treated with geneFECTOR (GF) alone or transfected with control (CTL) or HO-1 siRNA (seq2) were labelled with 5-chloromethylfluorescein diacetate (6.25 μM) and seeded onto gelatin-coated 96-well plates in the absence (UT) or the presence of VEGF for 40 min. Fluorescence was measured pre- and post-washing, with adhesion expressed as the percentage of bound cells relative to the total number seeded. ( G ). HUVEC were left untreated or transfected with control or vimentin siRNAs. Cells were then left untreated (Control) or infected with Ad0 and AdHO-1 for 24 hrs. Cell proliferation was quantified by BrdU ELISA. Data are presented as mean ± SEM (n = 3–5 experiments), * p < 0.05, *** p < 0.001.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Transfection, Staining, Immunofluorescence, Confocal Microscopy, Fluorescence, Infection, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

doi: 10.1038/srep29417

Figure Lengend Snippet: VEGF treatment of endothelial cells activates HO-1-dependent migration, proliferation and angiogenesis in vitro and in vivo . Microarray and proteomic analyses revealed cyclin A1, cyclin E1 and vimentin as downstream targets of HO-1. VEGF induced an HO-1-dependent increase in cdk2 kinase and calpain activity. These changes increased cell cycle progression and vimentin cleavage respectively. The role of HO-1 was established using specific siRNAs and HO-1 agonists. Similarly the importance of both cyclin A1 and vimentin in pro-angiogenic effects of VEGF and HO-1 activity were demonstrated by cyclin A1 and vimentin gene silencing.

Article Snippet: Single sequence siRNA against human HO-1 (siHO-1 seq2) was purchased from Qiagen (Manchester, UK).

Techniques: Migration, In Vitro, In Vivo, Microarray, Activity Assay