Fig. 1. AIF deficiency enhances the formation of cytoplasmic SGs in response to HAsO₄. (A) Failure of AIF to modulate the expression of inducible HSP70 or HSP27. HeLa cells were subjected to siRNA specific for human AIF (siRNA AIF) or mouse AIF (siRNA Co) for 48 hours, followed by culture in the absence (Co) or presence of HAsO₄ (3 hours, 1 mM), extraction of total cellular proteins and immunoblot detection of AIF, HSP70, HSP27 and GAPDH. (B) Abundance of cytoplasmic SGs. Cells treated as in A were stained for the detection of AIF (green fluorescence), the SG protein TIA-1 (red fluorescence) or chromatin (blue fluorescence). Note the accumulation of TIA-1⁺ cytoplasmic granules in HAsO₄-treated, AIF-negative cells. The histogram illustrates the percentage (±s.d., n=5) of cells accumulating SGs in the cytoplasm. (C) Failure of AIF to stimulate the generation of nuclear SGs. Cells treated as in A were stained with antibodies specific for constituents of nuclear SGs (HSF1, red) and cytoplasmic SGs (TIA-1, green). As a positive control for the generation of nuclear SGs, cells were treated for 18 (instead of 3) hours with HAsO₄. Note that cytoplasmic SGs form in cells that lack nuclear SGs. Results are representative of five independent experiments yielding similar results.