Image analysis

Images were analyzed for total fluorescence, co-localization, puncta, and cell numbers, as appropriate, using Imaris Bitplane and ImageJ image analysis software. For quantification of stress granules, PABP and TIA1 co-labeled sections were thresholded (TIA1 fluorescence intensity above 69.6, PABP intensity above 9.7) with nuclear masking using DAPI. The number of cytoplasmic (defined by exclusion from DAPI-positive nuclei) puncta with diameter greater than or equal to 2 μm co-positive for both TIA1 and PABP were counted and divided by the total number of DAPI-positive nuclei per image. For quantification of nuclear versus cytoplasmic immunofluorescence, images were processed as described above, and the total immunofluorescence of TIA1 within (nuclear) and outside (cytoplasmic) of DAPI-positive nuclei was calculated, and divided by the total number of DAPI-positive cells per image. Statistical analysis was performed using GraphPad Prism data analysis software.