FIGURE 6.

Transcript length and transcriptional state influence mRNA recruitment into ER-associated stress granules following arsenite stress. Representative smFISH of long transcript size-paired genes GRP94 (magenta, 2412 nt) and NCL (red, 2133 nt), and short transcript size-paired genes CCN2 (yellow, 1050 nt) and GAPDH (green, 1008 nt) mRNAs in untreated and arsenite-treated HeLa cell cultures. Where indicated, cell cultures were treated with ActD or triptolide in addition to arsenite. To evaluate the ER-association and presence of the SG protein marker G3BP1 in observed RNA granules, cells were permeabilized in digitonin-supplemented buffers to extract cytosolic contents while leaving the ER membrane intact, and costained for G3BP1 by immunofluorescence. Dotted boxes indicate regions of grayscale insets for mRNA and G3BP1 protein channels, as well as color merge, for digitonin permeabilized cells (below). DAPI nuclear stain (blue) included in all images. Full cell scale bars = 20 µm, inset scale bars = 4 µm.