FIG. 2.

Knockdown of GPx4 in mouse NIH 3T3 cells, growth rates, and morphology. (A) NIH 3T3 cells were stably transfected with the pU6-m3 or siGPx4 (siGPx4-2 and siGPx4-3) constructs and the expression of GPx4 protein and mRNA examined by labeling cells with ⁷⁵Se (upper panels, visualized with a PhosphorImager) or by Northern blotting (middle panels), respectively. The effects of knocking down GPx4 in the two siRNA-transfected cell lines were examined separately and are shown in separate panels: left panel, siGPx4-2, and right panel, siGPx4-3. Extracts were analyzed from NIH 3T3 cells (lanes 1 and 4); pU6-m3 cells (lanes 2 and 5); siGPx4-2 cells (lane 3), and siGPx4-3 cells (lane 6). 18S and 28S ribosomal RNAs are shown in lower panels and their levels were assessed to monitor sample loading and to assess levels of GPx4 mRNA. Protein molecular weight markers are shown on the left and selenoproteins (or GPx4 mRNA) on the right side of each panel. (B, C) NIH 3T3 cells stably transfected with pU6-m3 or siGPx4-3 were seeded at a density of 2 × 10⁵ cells/60 mm culture dish and grown as given in Materials and Methods. In (B), growth rates of both cell lines were determined by counting cell numbers at 24, 48, and 72 h; *p < 0.02; NIH/siGPx4-3 was compared with NIH/pU6-m3 and in (C), cell morphology was examined at 48 h and photographed with an inverted phase contrast microscope.