Marcia Field
Dołączył: 16 Gru 2019 Posty: 3
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Wysłany: Pon Gru 16, 2019 06:56 Temat postu: puma fierce |
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ÿþSimilar to the PUMA down-regulation, over-expression of EGFRvIII in puma fierce U87MG cells resulted in significant resistance to anisomycin-induced apoptosis ( Fig. 3c,d ). After treatment with anisomycin, there was only 3.1% apoptosis in U87MG-EGFRvIII cells. In contrast, the isogenic low EGFR expressing U87MG-vector cells underwent massive apoptosis (49.8%), similar to the level observed with PUMA down-regulation (45.5%). The results in Fig.
PUMA is known to localize on the mitochondrial membranes to initiate apoptosis upon appropriate stress [ 40 ; 41 ]. Rationalized by our data showing that EGFR and EGFRvIII co-express and interact with PUMA and that these two pathways are rihanna puma creepers inversely linked to apoptosis, we examined whether EGFR/EGFRvIII might regulate PUMA sub-cellular localization. Thus, we analyzed the cytoplasmic/mitochondrial distribution of PUMA in the isogenic pair, U87MG-EGFRvIII and U87MG-vector cells.
In contrast, in U87MG-vector cells fenty puma slides with very low level of EGFR expression, PUMA was exclusively present in the mitochondrial fractions independent of apoptotic stress ( Fig. 5b ). Effectiveness of cell fractionation is indicated by the absence of the cytoplasmic marker, ±-tubulin, in the mitochondrial extracts and the absence of the mitochondrial marker, Cox IV, in the nonmitochondrial extracts.PUMA is sequestered in the cytoplasm of EGFR- and EGFRvIII-expressing GBM and breast puma ignite cancer cells.
The extent of PUMA mitochondrial translocalization, mtPUMA Index, is computed as described earlier in Materials and Methods. (A) PUMA is primarily localized in the cytoplasm of U87MG-EGFRvIII cells. U87MG-EGFRvIII cells treated with vehicle control, staurosporin (ST, 1 uM) or anisomycin (AN, 100 ng/ml) were harvested and fractionated into mitochondrial and non-mitochondrial fractions. Protein extracts from both fractions were subjected to western blotting to detect PUMA.
Fig. 6a (left panel) shows that the ability of EGFR to bind to PUMA was similar in U87MG-EGFR cells with and without EGF stimultion following serum starvation. As indicated by the absence of auto-phosphorylated EGFR black puma (p-EGFR, Y1068), serum-starved U87MG-EGFR cells express inactive EGFR ( Fig. 6a-right ). In contrast, p-EGFR is readily detected in EGF-treated cells, indicating that EGF efficiently activated EGFR in these cells.
U87MG-EGFR cells were serum-starved for 24 hrs and stimulated with EGF for 20 mins. The cells were harvested and subjected to immunoprecipitation/western blotting (left panel) and western blotting (right panel). An EGFR antibody was used to immunoprecipitate EGFR. Control IgG did not yield signals indicating specificity. Auto-phosphorylated Y1068 residue serves as an indicator for EGF-induced EGFR activation. (B) EGFR kinase [img]http://www.czcrush.com/images/detail/black puma-579iiy.jpg[/img] activity is not required for their interaction with PUMA. |
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