Oxidative Medicine and Cellular Longevity / 2015 / Article / Fig 5

Research Article

Quercetin Affects Hsp70/IRE1α Mediated Protection from Death Induced by Endoplasmic Reticulum Stress

Figure 5

Hsp70-IRE1α axis protects from apoptosis. (a) Q induces fragmentation of PARP-1. Western blot was performed with anti-PARP-1 followed by peroxidase-conjugated secondary antibody to analyze this protein (FL = full length; CL = cleaved) in the lysates of untreated or Q (10 μM) pretreated cells and thereafter treated with none or TN for 24 h. Western blot of Hsp90 is shown at the bottom as a loading control. Representative blots are shown. (b) Western blot analysis of PARP-1 (FL = full length; CL = cleaved) by specific antibody in the lysates of U937 cells transfected with equal amounts of Hsp70 siRNA or scr-siRNA (Santa Cruz) for 72 h and exposed to none or to TN 1 μM for further 24 h. Western blot analysis of Hsp90 is shown at the bottom, as a loading control and also to confirm the specificity of the transfected siRNA. Representative blots are shown. (c) Detection of cell death by evaluation of sub-G1 events. U937 cells were pretreated for 60 min with none or z-VAD.fmk (70 μM), then for further 30 min with none or Q (10 μM) or 4μ8c (10 μM), and thereafter for 24 h with none or TN (1 μM). The cells were fixed and stained with PI to evaluate sub-G1 events in the cell cycle by cytofluorimetric analysis performed on ≥10000 events. The values reported are means ± S.D. (). Assessment of cell death showed statistically significant differences between the data obtained in the cultures treated with TN and Q and z-VAD in comparison with the cultures treated only with TN and Q () or between the data obtained in the cultures treated with TN and 4μ8c and z-VAD in comparison with the cultures treated only with TN and 4μ8c ().
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