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. 2021 Mar 19;41:101947. doi: 10.1016/j.redox.2021.101947

Fig. 7.

Fig. 7

NOX4 promotes ferroptosis by oxidative stress-induced lipid peroxidation in human astrocytes.

(A) Representative immunofluorescence images of 4-HNE expression in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing 4-HNE (red) (n = 10 per group). DAPI-stained nuclei are shown in blue. The shape of cells showed shrinkage and lipid peroxidation-derived droplets in the plasma membrane were indicated (white arrows). Symbols expressed by white dotted line indicate the shape of cells. Scale bars, 20 μm. (B) Quantification of 4-HNE positive astrocytes from immunofluorescence images in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Representative immunoblot analysis for 4-HNE and MDA protein levels (left) and quantification for 4-HNE and MDA protein levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. *p < 0.05 using the two-tailed Student's t-test. (D) Quantification of iron levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± standard deviation (SD). *, p < 0.05 by Student's two-tailed t-test. (E) Representative 3D images of control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 images per group). The morphological features of cytotoxicity were indicated (white arrows). Scale bars, 20 μm. (F) Quantification of the morphological dead cells in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in total 100 cells in 10 individual images per group). Data are mean ± SD. **, p < 0.01 using the two-tailed Student's t-test. (G) Cytotoxicity assay in control (Control) and NOX4 overexpressing (NOX4) human astrocytes was determined by lactate dehydrogenase (LDH) levels. Data are representatives of three independent experiments. Each experiment was done in triplicate. Data are mean ± SD. **, p < 0.01 using two-tailed Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)