Quantification of stress granule formation.

Control or PKR-deficient HeLa cells were transfected with either a green fluorescent protein (GFP) or pTRS1 expression vector. Twenty-four hours after transfection, cells were treated with arsenite for 1 h and then processed for immunofluorescence microscopy as above. Transfected cells (i.e., GFP or pTRS1 positive) were identified, and the relative fluorescence intensity of the GFP or pTRS1 signal in each cell was quantified using ImageJ software. The relative pTRS1 or GFP expression level in each cell was scored on a scale of 1 to 100, with 100 representing the highest observed expression. The presence of stress granules in each transfected cell was then determined by measuring the formation of G3BP1 puncta. Transfected cells containing two or more G3BP1 puncta were considered positive for stress granules, while those with fewer than two puncta were considered negative. The range of fluorescence intensity for GFP and pTRS1 was divided into quartiles (1 to 25% of maximum, 26 to 50% of maximum, etc.), and the percentage of cells containing stress granules within each expression quartile was calculated. A minimum of 100 GFP- or pTRS1-positive cells were analyzed. The data are displayed as the percent transfected cells containing stress granules within each quartile of GFP or pTRS1 expression.