Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 May 13;184(10):2649–2664.e18. doi: 10.1016/j.cell.2021.03.031

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2021 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Figure S1 — 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.