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Cells than in HepG2 cells (Fig. 4B, {top|leading|best|prime
Representative results from certainly one of 3 independent SW033291Biological Activity transfection experiments are shown.VOL. Intensity values from Northern blotting of HBV RNA had been normalized to -actin. Representative final results from among 3 independent transfection experiments are shown.VOL. 79,HBx BINDS DDB1 TO Market HBV REPLICATIONFIG. four. HBx promotes HBV replication and induces cell death via distinct DDB1-dependent pathways. (A) Human hepatoma HepG2 and Huh-7 cells had been transfected with wild-type HBV or HBV( X) genomic DNA with each other with a GFP gene to assess transfection efficiencies by FACS evaluation. The volume of core particle-associated HBV DNA replicative intermediates was assessed by Southern blot analysis three days just after transfection as described in the legend to Fig. 1. Transfection efficiencies were equivalent for the two genomic constructs in every single cell line, but crucial differences were noticed between the two cell lines ( 5 in HepG2 cells versus 20 in Huh-7 cells [data not shown]). Therefore, the amounts of sample analyzed were corrected accordingly. Among two independent transfection experiments is shown. The single-stranded (ssDNA) and double-stranded (dsDNA) HBV DNA replicative forms are indicated around the correct. (B) Western blot evaluation. Whole-cell extracts ready from HepG2 or Huh-7 cells transfected with GFP-HBx or empty vector (vect) (major) or from the indicated untransfected cell lines (bottom) have been separated by gel electrophoresis. Immunoblot analyses were performed with antibodies to HBx (major), DDB1 (bottom), and, as a control for loading, -tubulin. In the upper gel, fourfold-larger amounts of HepG2 protein extract were loaded on the gel to appropriate for transfection efficiencies. (C) Clonogenic cell survival assay. HeLa, Huh-7, and HepG2 cells were either mock transduced (Mock) or transduced with lentivirus vectors expressing the indicated GFP-HBx fusion proteins. Transduction efficiencies were comparable, as assessed by FACS analysis (data not shown). Cells have been PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20854184 then seeded at acceptable dilutions in six-well culture dishes. Right after 16 days of undisturbed growth at 37 , the surviving cells were fixed and stained with crystal violet. (D) Huh-7 and HepG2 cells were transfected having a GFP-expressing plasmid bearing a hygromycin resistance gene either alone (vect) or with each other with equal amounts in the indicated HBV genomic DNA. The transfected cells were seeded at acceptable dilutions within a six-well culture dish and cultured in medium containing hygromycin. Drug-resistant colonies have been fixed and stained with crystal violet 20 (HepG2) or 15 (Huh-7) days after transfection. Note that the HBV replication assay presented in panel A was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26891946 performed three days just after transfection, at which time the HBx-expressing HepG2 and Huh-7 cells do not show any in the clear alterations in morphology that typically precede HBx-mediated cell death (information not shown).cells is unlikely to be as a consequence of protein instability. These outcomes lead us to propose that HBx stimulates HBV replication by way of its association with DDB1 by way of a pathway that differs at some point from that leading to cell death. DISCUSSION The HBx protein has been reported to exhibit several different various activities in tissue culture cells, including induction of cell death and stimulat.
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