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Rm. c) Specific RT-PCR amplification of hHIF2 sequence was used to
f) Western blot evaluation of HIF1 levels revealed no variation between the CS-2804 web different cell clones in both 21 and 4 O2. fected cells (NT) or cells CS-3168 chemical information transfected with the empty pCDNA3.1 get 112648-68-7 vector were used as controls (C). To confirm the activity of HIF2 and HIF2(1?85) exogenous proteins, we studied the expression of tyrosine hydroxylase (TH), a known HIF2 target gene in the Amlexanox sympathetic nervous system [17]. We observed an increase in TH protein expression in HIF2 clones, and a reduction in HIF2(1?85) compared to control cells (Fig. 1d). We next incubated the cells in normoxia (21 O2) or in low oxygen tension (4 O2) for 6 h and evaluated the release of VEGF by ELISA (Fig. 1e). In controls cells, we confirmed that VEGF expression was indeed induced, although moderately, in response to decreased oxygen tension (p = 0,04). As expected, we noted a marked increase in VEGF levels in both E1 and E2 clones, in normoxia (161 (p = 0,017) and 178 (p = 0,012) increase respectively as compared to control cells) and in 4 O2 (117 (p = 0,004) and 272 (p < 0,001) increase respectively). In contrast, VEGF protein concentration was not modified in DN5 and DN8 clones in normoxia compared to control cells, while its hypoxic induction was repressed in both clones. Western blot evaluation of HIF1 protein levels confirmed that the differences observed were not due to a deregulation of HIF1 protein expression, but attributable to HIF2 transfections (Fig. 1f).Page 4 of(page number not for citation purposes)BMC Cancer 2007, 7:http://www.biomedcentral.com/1471-2407/7/Effect of HIF2 on neuroblastoma cell phenotype Hypoxia induces an immature phenotype of neuroblastoma cells [11], while inhibition of the HIF pathway by VHL overexpression mediates differentiation toward a neuron-like morphology [12]. We searched for such modifications in HIF2 and HIF2(1?85) clones, but could not detect any change in the typical cell shape nor in the number of neuronal extension (Fig. 2a). To confirm this observation, we studied the expression of Id2 and CgA markers, which are respectively induced and downregulated during hypoxia-mediated neuroblastoma dedifferentiation [11,18]. Such RT-PCR analyses revealed the absence of any noticeable difference in their expression between the various clones (Fig. 2b).Unexpectedly, we observed a marked modification in N1E-115 cell size (Fig. 2a and 2c): both HIF2 clones displayed an average 30 increase in cell diameter (p < 0.001), while HIF2(1?85) cells were 15 to 25 smaller than native or pCDNA3.1-transfected cells (p < 0.001). We performed Western blot experiments to detect the expression and the phosphorylation of two protein kinases, known to play a role in the modulation of cell size [19], i.e. Akt (Fig.Rm. c) Specific RT-PCR amplification of hHIF2 sequence was used to confirm the efficiency of transfection with both hHIF2 (E1 and E2) and hHIF2(1?85) (DN5 and DN8) expressing vectors. Western blot using a specific anti-human HIF2 antibody confirmed the nuclear presence of the wild-type protein in E1 and E2 clones. d) Tyrosine hydroxylase (TH) expression was assessed by western blot and e) VEGF protein expression quantified by ELISA in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14575864 duplicate 21 or 4 O2 atmosphere.
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