Actinomycin D inhibits RNA binding protein granulation during the UPR. (A) Representative GRP94 smFISH (magenta) with immunofluorescence costaining for HuR (cyan) in control (untreated or ActD) and stressed (treatment with DTT, with and without ActD) HeLa cell cultures. Note redistribution of HuR from the nucleoplasm to the cytoplasm in response to ActD treatment. Boxes indicate regions of grayscale insets for mRNA and protein channels, as well as color merge, for each condition (right). (B) As in A but immunofluorescence staining for G3BP1 (cyan). (C) As in A but immunofluorescence staining for PABP (cyan). (D) Representative GRP94 smFISH (magenta) with immunofluorescence costaining for PABP (cyan) in cells treated with arsenite with and without cycloheximide (CHX). Boxes indicate regions of grayscale insets for mRNA and protein channels, as well as color merge, for each condition (right). (E) As in D but with DTT stress. (F) Representative GRP94 smFISH (magenta) with immunofluorescence costaining for G3BP1 (cyan) in cells treated with arsenite with and without ActD. Boxes indicate regions of grayscale insets for mRNA and protein channels, as well as color merge, for each condition (right). DAPI nuclear stain (blue) is included in all images. Full cell scale bar = 20 µm, inset scale bar = 4 µm.
