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. 2021 Sep 16;184(19):4886–4903.e21. doi: 10.1016/j.cell.2021.08.001

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© 2021 The Author(s)

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

PMC Copyright notice

Figure S2 — MCP-based synthetic film-like organelles enrich tRNA^Pyl, mRNA::ms2, and cellular ribosomes, related to Figure 4

(A–B) Images used for the 3D rendering in Figure 4. HEK293T cells expressing the indicated OT organelle, tRNA^Pyl, and a construct encoding NLS::EGFP^39TAG::ms2 in the presence of the ncAA SCO-K. All images show a selected plane of a z stack.

(A) Corresponding to Figure 4A. IF staining against PylRS and FISH staining of tRNA^Pyl show strong tRNA^Pyl recruitment. Top to bottom: PylRS (magenta), tRNA^Pyl (yellow), merged (NLS::EGFP^39TAG::ms2 shown in green).

(B) Corresponding to Figure 4C. IF staining against PylRS and RPL26L1 show that endogenous ribosomes are recruited into OT film-like organelles. Note that owing to the abundance of ribosomes, a substantial staining outside of the OT organelles can be observed. Top to bottom: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP^39TAG::ms2 in green). Scale bars: 20 μm.

(C) IF staining against PylRS and FISH staining of mRNA::ms2 show recruitment of ms2-tagged mRNA into membrane-associated OT organelles. Top to bottom: PylRS (magenta), mRNA::ms2 (yellow), merged (NLS::EGFP^39TAG::ms2 in green).