UPR activation elicits ER-associated stress granules. (A) Representative GRP94 smFISH (magenta) with immunofluorescence costaining for the stress granule protein markers HuR, G3BP1, and PABP (cyan) in untreated and DTT-treated HeLa cell cultures. Dotted boxes indicate regions of grayscale insets for mRNA and protein channels, as well as color merge, for DTT-treated cells (right). (B) Representative GRP94 smFISH (magenta) and G3BP1 immunofluorescence (cyan) costaining in DTT-treated cells. Following DTT treatment, cells were permeabilized with digitonin-supplemented buffer to release cytosolic contents (digitonin) or sequentially treated with digitonin and n-dodecyl-β-d-maltoside (DDM) buffers to solubilize organelle membranes. See also Supplemental Figure S1 for detergent permeabilization protocol validation. (C) As in B but with arsenite stress. (D) Representative micrographs of ER membrane protein TRAPα immunofluorescence (yellow) with GRP94 smFISH (magenta) and/or G3BP1 immunofluorescence (cyan) costaining in unfractionated and cytosol-depleted (digitonin-permeabilized) cells following DTT treatment or untreated control. Boxes indicate regions of interest for (E). (E) 3D renderings of representative granules from (D) in unfractionated (Supplemental Movie 1) and digitonin-permeabilized (Supplemental Movie 2) cells. DAPI staining (blue) is indicated for all images. Full cell scale bars = 20 µm, inset and 3D scale bars = 4 µm.
