Knocking down ATF4 in NSC34 cells alters the redox state of the ER, contributing to mutant SOD1 aggregation.

<p>(<b>A</b>) ROS levels were determined in shATF4 and shLuc NSC34 cells at basal levels or after treatment with 1 μg/ml Tm for 16 h using dichlorofluorescein (DCF) staining and FACS analysis. Right panel: Quantification of mean DCF fluorescence. (<b>B</b>) The generation of H₂O₂ inside the ER was determined in shATF4 and shLuc cells after transient expression of HyPer-ER_lum construct. After 72 h, cells were treated or not with 1 μg/ml Tm for 6 h and fluorescence emission determined by live cell-imaging microscopy. (<b>C</b>) NSC34 shATF4 cells were transiently transfected with SOD1^G85R-EGFP plasmid and treated with 400 mM trolox after 24 h. Two days after mutant SOD1 aggregation was analyzed by Western blot. Left panel: quantification of SOD1 aggregates. (<b>D</b>) NSC34 shATF4 cells were co-transfected with expression vectors for SOD1^G85R-EGFP together with ERO1Lα plasmid or empty vector. Left panel: Then, mutant SOD1 aggregation was monitored after 72 h by Western blot (left panel, Bar 50 μm). Right panel: In parallel, mutant SOD1 inclusions were visualized by fluorescent microscopy and quantified. (<b>H</b>) Control and shATF4 NSC34 cells were exposed to 5 μg/ml Tm for 16 h and cell death was detected after propidium iodide (PI) staining, and visualized with a fluorescent microscope. As a positive control, cells were treated for 16 h with 20 μm etoposide (Eto). Data is representative of three independent experiments. Arrows heads shows PI stained cells. Ph C: Phase Contrast. In all plots, mean and standard deviation is presented of three independent experiments. <i>p</i> value was calculated with Students t-test, *: <i>p</i><0.05.