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Fig. 6. vFLIP rescue of MVECs from anoikis requires activation of NF- B and is partly attributable to a secreted survival factor(s). (A) Human dermal MVECs were either left untreated or were pretreated with 20 µM Bay 11-7082 for 1 hour. Untreated cells, and cells incubated with Bay 11-7082, were then washed twice with HBSS and either not transduced (labelled `Uninfected') or transduced with a lentiviral vector encoding GFP alone or vFLIP and GFP. 48 hours post transduction, cells were detached from the matrix by trypsinization and either plated on control wells, or anoikis was induced by plating cells on polyHEMA (PH)-coated wells for 16 hours. Apoptosis was then measured using a cell death detection ELISA kit that measures DNA fragmentation. The results shown are the mean values from three independent experiments, with error bars indicating the standard deviation (s.d.). The transduction efficiency was assessed by FACScan analysis of cells expressing GFP. 57% of MVECs infected with the vFLIP lentivirus were GFP positive, and 73% of MVECs infected with the GFP lentivirus were GFP positive. (B) Human dermal MVECs were either not transduced (labelled `Uninfected' or `UI' on the graph) or transduced with a lentiviral vector encoding GFP alone or vFLIP and GFP. 48 hours post transduction, cells were detached from the matrix by trypsinization and either plated on control wells or anoikis was induced by plating cells on PH-coated wells for 16 hours. A subset of cells from all three groups was washed post trypsinisation and plated in 500 µl of supernatant, diluted 1:2 in normal medium, from untransduced, GFP- and vFLIP-transduced cells on PH-coated wells for 16 hours to assess the effect of secreted factors in the respective supernatants on the survival of MVECs upon detachment. Apoptosis was measured using a cell death detection ELISA kit that measures DNA fragmentation. The results shown are the mean values from three independent experiments, with error bars indicating the s.d. Differences were calculated using paired Student's t tests, in which P<0.05 was considered significant. *P<0.05; **P<0.01. Transduction efficiency was assessed by FACScan analysis of cells expressing GFP. 63% of MVECs infected with the vFLIP lentivirus were GFP positive, and 75% of MVECs infected with the GFP lentivirus were GFP positive.
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, packaging signal; RRE, rev-responsive element; cPPT, central polypurine tract; SFFV, spleen focus-forming virus promoter; WPRE, wood chuck posttranscriptional regulatory element; Ub, ubiquitin promoter; 
U3, deletion in U3 region (SIN vector). (B) Human dermal MVECs were either not transduced or transduced with a lentiviral vector encoding GFP alone or vFLIP and emerald GFP at a MOI of 30. 48 hours post transduction, the transduction efficiency was assessed by FACScan analysis. Live cells were gated (not shown) and expression of GFP was analysed using CellQuest software. 10,000 live cells were analysed per sample. The mean percentage of transduced MVECs was 55% for vFLIP_eGFP (s.d.=5.06) and 76.3% for GFP (s.d.=4.33) determined from three experiments. Values below the percentage transduced cells represent the mean fluorescence intensity (MFI) of the positive cells. (C) GFP expression and cell morphology measured using a BioRad confocal microscope 48 hours post transduction with the two vectors at a MOI of 
10.
