Fig 2: Cell biological and biophysical properties of EML4-ALK membraneless cytoplasmic protein granules, related to Figure 1(A) Anti-FLAG immunofluorescence of FLAG-tagged EML4-ALK expressed in human epithelial cell line Beas2B. DAPI serves as a nuclear stain. Image is representative of at least 75 analyzed cells in total over 3 independent experiments. Scale bar = 5 μM.(B) Live-cell confocal imaging of human epithelial cell line Beas2B upon expression of mTagBFP2::EML4-ALK. Image is representative of 200 analyzed cells in total over 5 independent experiments. Scale bar = 5 μM.(C) Live-cell confocal imaging of human epithelial cell line Beas2B upon expression of mTagBFP2::EML4-ALK and mEGFP-tagged organelle markers as listed. Membrane dye experiments were conducted using live cells incubated with CellTracker CM-DiI Dye (Invitrogen). Each panel is a representative image of at least 20 analyzed cells per condition in 3 independent experiments. Scale bar = 5 μM.(D, E) Subcellular fractionation by ultracentrifugation ± detergent (1% Triton X-100) to disrupt lipid membranes in STE-1 (D), an EML4-ALK expressing cancer cell line, and Beas2B cells expressing EML4-ALK (E). In both cell lines, EML4-ALK and DCP1B are statistically distinct from the lipid membrane-associated proteins, which shift from the insoluble fraction (pellet) to the soluble fraction with detergent (p < 0.05 for all comparisons by one-way ANOVA with post hoc Tukey’s HSD test, except for DCP1B versus EEA1 in panel D and E and ALK versus EEA1 in panel E). Bar graphs reflect quantification of western blotting results for 3 independent replicates. Fraction in pellet calculated as ratio of the insoluble fraction to total (insoluble plus supernatant fractions) as assessed by western blotting. Error bars represent ± SEM.(F, G) Subcellular fractionation by ultracentrifugation ± RNase A treatment for 30 minutes in EML4-ALK expressing cancer cell line H3122. P-body protein DCP1B partially shifts from the insoluble (pellet) fraction to the supernatant (sup) upon RNase A treatment, in contrast to EML4-ALK. Western blotting images are representative of at least 5 independent experiments.Fraction in pellet (G) calculated as ratio of the insoluble fraction to total (insoluble plus supernatant fractions) as assessed by western blotting (F). DCP1B demonstrates a significant RNase-dependent reduction in the insoluble fraction (p < 0.05 by paired t test) compared to EML4-ALK.(H) Live-cell imaging of human epithelial cell line Beas2B co-expressing mTagBFP2::EML4-ALK and eGFP::DCP1B treated with 5% hexanediol (hex) and imaged at respective time points. Images are representative of at least 5 analyzed cells in 3 independent experiments. White arrows indicate a representative EML4-ALK cytoplasmic protein granule (multiple non-highlighted granules are also pictured in all panels). Quantification of granule persistence shown in Main Figure 1D.

Fig 3: Targeting RNase L to P‐bodies did not increase the effect of RNase L activation on P‐body number or volume Full‐length Dcp1 RNase L (DRL) fusion proteins are expressed. Western analysis using anti‐RNase L antibody of whole cell lysates from A549 (RL‐WT) cells, A549 RNase L knock out cells (RL‐KO), and RL‐KO cells transduced with lentiviral vectors containing either wild‐type RNase L fused to DCP1a (RL‐KO DRL‐WT) or catalytic mutant RNase L‐R667A fused to DCP1a (RL‐KO DRL‐CM). Arrows on left indicate migration of the endogenous RNase L (RL) and the DCP1 RNase L fusion proteins (DRL).DRL fusion proteins co‐localize with P‐bodies. IF analysis using anti‐DCP1b antibody to detect P‐bodies (DCP1) or anti‐Flag antibody to detect Flag‐tagged DCP1a‐RNase L fusion proteins. Scale bar 10 microns.DRL‐WT fusion protein is active. GAPDH smFISH and IF analysis using anti‐G3BP antibody (G3BP) and anti‐Dcp1b antibody (DCP1) in A549 RL‐KO cells with DRL‐WT or DRL‐CM fusion proteins either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 h. Scale bar 10 microns.Number of DCP1 foci per cell in mock‐ and PIC‐treated RL‐WT and RL‐KO cells expressing DRL‐WT relative to the number in mock‐treated cells. Wilcoxon Signed Rank test, ns, non‐significantAverage volume of individual DCP1 foci in mock‐ and PIC‐treated cells. One‐way ANOVA with Sidak’s multiple comparisons test. Data information: (D, E) Bar graphs show mean + SD for N = 4 independent experiments. ns, non‐significant. Source data are available online for this figure.

Fig 4: The number and size of P‐bodies is unaffected in cells with active RNase L GAPDH smFISH and IF analysis using anti‐G3BP antibody (G3BP) and anti‐Dcp1b antibody (DCP1) in A549 cells (RL‐WT) or A549 RL‐KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 h. Scale bar 10 microns.Graph of the fraction of G3BP foci with different volumes in PIC‐treated RL‐WT and RL‐KO cells.Number of DCP1 foci per cell in PIC‐treated RL‐WT and RL‐KO cells relative to the number in mock‐treated cells. Wilcoxon Signed Rank test, ns, non‐significant.Average volume of individual Dcp1 foci in mock‐ and PIC‐treated RL‐WT and RL‐KO cells. One‐way ANOVA with Sidak’s multiple comparisons test, ns, non‐significant. Data information: (C, D) Bar graphs show mean + SD for N = 4 independent experiments. Source data are available online for this figure.

Fig 5: RNase L inhibits SG but not PB assembly and promotes RLB assembly. A, Western blotting analysis of PKR and RNase L in parental (WT), RNase L–KO (RL-KO), PKR-KO, and RNase L/PKR double knockout (RL/PKR-KO) A549 cells. B, IF for G3BP1 (SG marker) and DCP1b (PB marker) in the indicated A549 cell lines transfected with or without poly(I·C). To calculate the percentage of cells with G3BP1+ foci (RLBs or SGs), 25–195 cells from between 3 and 15 fields of view were analyzed. C, histogram of the number of G3BP1-positive foci binned by area as represented in B. D, histogram of the number of DCP1b-positive foci binned by area as represented in B. E, similar to B but depicting RLB and PB association. F, IF for G3BP1 and FISH for poly(A)+ RNA in WT cells 4 h post-poly(I·C).