Figure 3. ISRIB addition rapidly dissolves pre-formed stress granules in live cells restoring translation.
(A) Live cell imaging of stress granules in U2OS cells stably expressing G3BP-GFP (SG marker). Cells were treated with 200 nM Tg for 40 min, 250 μM Ars for 30 min, 100 nM Pat A for 30 min, or 300 nM Hipp in the presence or absence of 200 nM ISRIB. Cells were imaged using an epifluorescence microscope. Representative images of at least two biological replicates are shown. (B) Quantitation of the percentage of cells containing stress granules in the different conditions described in A. Images were collected from at least two independent experiments and the number of cells with SGs or no SGs counted. The number of cells analyzed for each condition was (sum of replicates): Control (N = 98), ISRIB (N = 81), Tg (N = 101), Tg + ISRIB (N = 84), Ars (N = 80), Ars + ISRIB (N = 55), Pat A (N = 58), Pat A + ISRIB (N = 50), Hipp (N = 41) and Hipp + ISRIB (N = 52). p-values are derived from a Student's t-test, *p < 0.05. (C) Stress granules were pre-formed with Tg for 40 min (as in Figure 3A) and then CHX (50 μg/ml) or ISRIB (200 nM) was added to the well, incubated for 10 min and images were collected. Representative images of at least two biological replicates are shown. (D) ISRIB quickly restores mRNA translation upon disassembly of stress granules. Cells were treated as in C with 200 nM Tg for 40 min and then DMSO, CHX (50 μg/ml), or ISRIB (200 nM) was added at the same time as [35S]-methionine. Cells were lysed after 15 min, protein was run in an SDS-PAGE gel and radioactivity was measured in each lane (N = 2, mean ± SD). (E) ISRIB quickly dissolves stress granules but does not affect P-bodies. Live cell imaging of U2OS cells stably expressing G3BP-GFP (SG marker) and Dcp1-RFP (P-body marker). Cells were treated with 200 nM Tg for 45 min followed by addition of 200 nM ISRIB at t = 0 min to the well and then imaged using spinning disk confocal microscopy. Images were collected every 30 s. The red arrows point to two representative P-bodies. Representative images of at least three biological replicates are shown.
Figure 3—figure supplement 1. ISRIB dose response and inactive analog in stress granule assay.
Figure 3—figure supplement 2. Representative SDS-PAGE gel of [35S]-methionine pulse as described in Figure 3D.
