FIG. 5.

Genetic silencing of beclin-1 prevents H₂O₂ toxicity in SH-SY5Y cells. (A) Western blotting showing the knock down (> 90%) of beclin-1 elicited by the specific siRNA (siBeclin). The filter was stripped and probed for actin as marker of protein loading. Similar results were obtained in two other experiments. Statistic of densitometry is reported. (B) Sham- and beclin-1–specific siRNA-transfected (siBeclin) cells adherent on coverslips were exposed or not to H₂O₂ for 15 min and then fixed and fluorescently double stained for beclin-1 and Golgin-97. Images (representative of three separate experiments) show that H₂O₂ induces the rapid polarization of beclin-1–positive aggregates in a Golgin-97–positive area. The experiment also confirms the efficient silencing of beclin-1 expression attained by the specific siRNA. Quantification of beclin aggregates–positive cells is indicated. (C) The extent of apoptotic cell death was assayed by flow cytometry analysis of annexin V-FITC positivity in sham- and beclin-1–specific siRNA-transfected cells exposed for 2 h to H₂O₂. Data from three independent experiments demonstrate that posttranscriptional silencing of beclin-1 protects the cells from H₂O₂ toxicity. Statistical significance between relevant treatments is indicated. (D) Fluorescence staining of bax and DAPI in sham- and beclin-1–specific siRNA-transfected (siBeclin) cells exposed or not to H₂O₂ for 120 min. Images (representative of three separate experiments) show that H₂O₂ induces bax activation and oligomerization in sham-transfected (∼50% of the population remained attached) not in siBeclin-transfected cells. Bar = 10 μm.