Oxidative Medicine and Cellular Longevity / 2015 / Article / Fig 2

Research Article

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

Figure 2

Hsp70 promotes survival of ER stressed cells. (a) Hsp70 was detected by western blot in the lysates of U937 cells either exposed or not to Q (10 μM, 30 min) and thereafter to none or TN 1 μM or TG 200 nM for 24 h. Blotted proteins were probed with anti-Hsp70 followed by peroxidase-conjugated secondary antibody. Western blot of β-actin is shown at the bottom as a loading control. Representative blots are shown. Densitometric quantification of the bands is shown at the bottom of the relevant lines as the ratio of Hsp70 in each line to the Hsp70 value observed in the lysate of untreated U937 cells. (b and c) Detection of cell death by evaluation of PI+ cells (b) or of sub-G1 events (c). U937 cells were pretreated for 30 min with none or Q (10 μM) or PES (10 μM) and then with none or TN (1 μM) or TG (200 nM). A portion of cells were unfixed and stained with PI (b) or fixed and stained with PI to evaluate sub-G1 events in the cell cycle (c) under cytofluorimetry. Both types of investigation were 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 or TG together with Q or with PES, in comparison with the cultures treated only with Q () or only with PES () or only with TN or TG (; ; ). (d) Western blot analysis of Hsp70 by specific antibody in the lysates of U937 cells transfected with equal amounts of Hsp70 siRNA or scr-siRNA for 72 h and exposed to none or to TN 1 μM for further 24 h. Western blot analysis of β-actin is shown at the bottom, as a loading control and also to confirm the specificity of the transfected siRNA. Representative blots are shown. Densitometric quantification of the bands is shown at the bottom of the relevant lines as the ratio of Hsp70 in each line to the Hsp70 value observed in the lysate of untreated U937 cells transfected with scrambled siRNA. (e) U937 cells transfected with Hsp70 siRNA or scr-siRNA for 72 h and exposed or not to TN 1 μM for 24 h were fixed with ethanol, stained with PI, and analyzed under cytofluorimetry to detect sub-G1 events in the cell cycle. Here are shown the mean ± S.D. of four independent experiments. Assessment of cell death showed statistically significant differences between the data obtained in the cultures treated with scr-siRNA and TN in comparison with the cultures treated only with scr-siRNA () or with Hsp70 siRNA and TN in comparison with scr-siRNA or Hsp70 siRNA ().
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