a, Oxidative stress and heat shock induce stress-granule formation in mouse ES cells. Stress-granule formation has not been extensively characterized in mouse ES cells. We therefore wanted to ensure that stress granule composition is the same in mouse ES cells compared to other cell types in which stress granules are more frequently studied. To test mouse ES stress granules, we stained with additional markers. Co-immunostaining with ATXN2 (green) and G3BP1 (red) after arsenite treatment (0.5 mM for 1 h) and heat shock (42 °C for 30 min) in mES cells showed clear labelling of stress granules. The overlay panel shows ATXN2 and G3BP1 overlap (yellow). Thus, stress granules in mouse ES cells appear to have similar markers as stress granules in other cell types. The experiment was performed in triplicate. Scale bar, 10 μm. b, c, DF1 and DF3 proteins relocalize to stress granules after heat shock and oxidative stress. DF1, DF2 and DF3 have high sequence similarity and show similar phase-separation properties. We therefore wanted to determine whether all these proteins associate with stress granules. Co-immunostaining was performed in mES cells with DF1 (red) or DF3 (red) with TIAR (green) after arsenite treatment (0.5 mM for 1 h) or heat shock (42 °C for 30 min). Along with DF2 shown in Fig. 2, DF1 and DF3 relocalize to stress granules treatment as visualized by the co-localization with TIAR. Scale bar, 10 µm. These findings are consistent with previous proteomic datasets of stress granules. A P-body proteome dataset48 showed that DF2 was enriched in P-bodies. DF2 ranked 152 among 1,900 P-body-associated proteins by abundance. All DF proteins were identified in a group of around 300 stress-granule-enriched proteins in a proteomics study of stress granules49. In another study, in vivo proximity-dependent biotinylation (BioID)-labelling study of G3BP1 and other stress-granule markers showed interactions with all DF proteins50. Another APEX labelling study51 of G3BP1 showed that the YTHDFs are 3 of the top 42 G3BP1-interacting proteins in the stress-granule proteome. Overall, these studies suggest that DF proteins are commonly seen in stress granules, and may be highly abundant relative to other stress-granule components. The experiment was performed in triplicate. d–f, DF2 relocalizes to stress granules after arsenite treatment in numerous cell types. The focus of this experiment was to determine whether DF relocalization to stress granules is likely to be a universal feature of stress granules. We therefore tested DF localization to stress granules in multiple cell types. Shown is co-immunostaining of HEK293 cells (d), U2OS cells (e), and NIH3T3 cells (f) with DF2 (red) and TIAR (green) after arsenite treatment (0.5 mM for 1 h) and heat shock (42 °C for 30 min). The overlay panel shows DF2 in stress granules based on its overlap with TIAR (yellow). The experiment was performed in duplicate. Scale bar, 10 μm. g, Confirmation of CRISPR–Cas9 knock in of NeonGreen–DF2. A western blot of HEK293T shows endogenous expression of NeonGreen–DF2. Note, only one allele contains the knock-in construct, accounting for the presence of unmodified DF2 in cells. h, Arsenite stress induces the localization of NeonGreen–DF2 into stress granules. We wanted to determine whether the ability of DF2 to undergo phase separation in vitro could be actively observed in cells. Unstressed HEK293T cells expressing NeonGreen-tagged DF2 protein show a diffuse cytoplasmic fluorescent signal. Upon arsenite stress (0.5 mM, 1 h), NeonGreen–DF2 phase-separates into stress granules. This confirms the ability of NeonGreen–DF2 to undergo phase separation in cells in response to stress. The experiment was performed in triplicate. Scale bar, 10 μm. i, Relocalization of DF2 to the nucleus does not occur after various stresses in various cell types. Because DF2 has been reported to relocalize to the nucleus 2 h after heat shock13, we wanted to determine whether any nuclear relocalization occurs in our experiments, which were performed immediately after stress. The ‘Stress condition’ column indicates the type and length of stress applied. The ‘Cell type’ column indicates the type of cell that was stressed. The ‘DF2 in nucleus’ column denotes the number of cells that were found to have DF2 in the nucleus immediately after stress. The ‘Total cells’ column indicates the number of cells that were examined for DF2 nuclear relocalization in each experimental condition. In all conditions, there was no cell that showed nuclear DF2 localization. Thus, DF2 localization is primarily in cytosolic stress granules at the time at which the stress is terminated. DF2 was not observed to relocalize to the nucleus at any time point or after any stress, including the 2-h post-heat-shock conditions described previously13. j, DF2 relocalization to stress granules does not require new mRNA or protein synthesis. We wanted to know whether an increase in DF2 expression or new m6A formation could be required for the formation of stress granules after heat shock. To test this, we blocked protein synthesis with puromycin and blocked new transcription with actinomycin D. Actinomycin D blocks m6A formation because m6A formation occurs co-transcriptionally17,18. The fluorescence micrographs show DF2 immunostaining in HEK293T cells treated with DMSO (left), puromycin (10 μg ml−1, middle), and actinomycin D (2.5 μg ml−1, right) for 15 min before and during incubation at 42 °C for 30 min. The ability of DF2 to relocalize to stress granules when transcription (actinomycin D) and translation (puromycin) was arrested was assessed by immunofluorescence staining for DF2. In each case, the formation of stress granules was unaffected, indicating that no new protein synthesis or new methylation is required for stress-granule formation. The time course of stress-granule formation is rapid, making it unlikely that new protein synthesis or methylation is involved in stress-granule formation. Additionally, heat shock is normally associated with inhibited transcription and translation, which further suggests that new protein synthesis and RNA methylation is unlikely to occur in the time course of stress-granule formation. On the basis of all this data, we propose that stress granule formation probably utilizes pre-existing patterns of m6A seen in unstressed cells to mediate the formation of stress granules. The experiment was performed in duplicate. Scale bar, 10 µm. k, l, m6A levels are not significantly altered immediately after arsenite and heat-shock stress in NIH3T3 cells. We wanted to test whether m6A levels in mRNA transcripts were altered as a result of cellular stress. NIH3T3 cells were subjected to arsenite (0.5 mM, 1 h) or heat-shock stress (43 °C, 45 min) and total RNA was extracted immediately after stress treatment. Total RNA was further purified by poly(A) selection to specifically assay m6A levels in mRNA transcripts. TLC38 revealed that there was no significant increase in m6A levels within poly(A) mRNA immediately after either stress condition in three biological replicates (see l). This indicates that cellular stress does not induce an increase or decrease in m6A over the time frame examined. Experiments were performed in duplicate. Bar heights in l represent mean and error bars represent s.e.m. Three biological replicates (n = 3) were analysed in the control, and four biological replicates (n = 4) were analysed after heat-shock and arsenite stress. Stress m6A/(A+C+U) ratios were analysed with a two-sided Student’s t-test.