YB-1 kd impairs SG assembly and sensitizes cells to oxidative stress. (A–C) U2OS cells transfected with siControl or siYB-1 siRNAs were treated with vehicle alone, arsenite (0.5 mM), or H₂O₂ (0.5 mM) for 1 h and immunoblotted using anti–YB-1 or anti-GAPDH antibodies (A). SGs were detected by IF in arsenite- (B) or H₂O₂-treated (C) siControl or siYB-1 U2OS cells using the indicated antibodies. Slides were counterstained with DRAQ5 to detect nuclei. SGs were quantified using ImageJ software by counting cells containing SGs divided by total cells, and represented by bar graphs. (D) Arsenite (0.5 mM)-treated siControl or siYB-1 U2OS cells were subjected to in situ hybridization using 5-FAM-oligodT and counterstained with anti–YB-1 antibodies. SGs were quantified as in B. (E) U2OS cells transfected with siControl or siYB-1 siRNAs were treated with arsenite (0.5 mM) for 60 min, then placed in full media without arsenite for another 60 min. Cells were fixed at the indicated time points and subjected to IF using anti–TIA-1 antibodies. SGs were quantified as in B. (F) U2OS cells transfected with siControl or siYB-1 siRNAs were treated with arsenite (0.5 mM) or H₂O₂ (0.5 mM) for 5 h, and apoptosis was measured by Annexin V-FITC flow cytometry. (G) Cell lysates from F were subjected to immunoblotting using the indicated antibodies. Mean values ± SD (error bars) are shown for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.