FIG. 4.

Lipid peroxidation in GPx4 knockdown cells. NIH 3T3 cells were stably transfected with the pU6-m3, siGPx4-3, or siGPx1 constructs. (A, B) Stably transfected cells were cultured and the levels of the lipid peroxidation by-products, MDA or 4-HNE, measured as described in the Materials and Methods. In (A), 3 × 10⁶ cells/sample of both cell lines were analyzed by the MDA assay. *p < 0.01; NIH/siGPx4-3 was compared with NIH/pU6m3. In (B), 4-HNE modified proteins were detected in both cell lines by fluorescence microscopy using 4-HNE antibodies wherein blue indicates nuclei stained with DAPI and green indicates 4-HNE modified proteins. (C, D) Cells stably transfected with pU6-m3 or siGPx4 were transiently transfected with the GPx1 expression or corresponding control construct as indicated. The two cell lines were compared by examining (C) intracellular MDA production as determined above. *p < 0.03; NIH/siGPx4-3 transfected with control vector was compared with NIH/pU6m3 transfected with control vector, and (D) ubiquitinated protein production in each cell line as determined by western blot analysis. Lower panel shows GPx1 expression determined by Western blotting with anti-GPx1 antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).