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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 S5 — λ_N22-based synthetic organelles enrich tRNA^Pyl, mRNA::boxB, and cellular ribosomes, related to Figure 2C

HEK293T cells expressing the indicated OT organelle, tRNA^Pyl and a construct encoding EGFP^39TAG::boxB in presence of the ncAA SCO-K. All images show a selected plane of a z stack.

(A) IF staining against PylRS and FISH staining of tRNA^Pyl shows strong recruitment of tRNA^Pyl into OT organelles. Left to right: PylRS (magenta), tRNA^Pyl (yellow), merged (NLS::EGFP^39TAG::ms2 in green). Shown is a selected plane of a z stack. Scale bars: 20 μm.

(B) 3D reconstruction corresponding to panel A. Left to right: PylRS (magenta), tRNA^Pyl (yellow), merged (NLS::EGFP^39TAG::ms2 in green in all panels). See also Video S1 for the animated 3D reconstructions. Scale bars: 10 μm.

(C) IF staining against PylRS and RPL26L1 shows that endogenous ribosomes co-localize with membrane-associated OT organelles. Note that due to the abundance of ribosomes, a substantial staining outside of the OT organelles can be observed. Left to right: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP^39TAG::ms2 in green). Shown is a selected plane of a z stack. Scale bars: 20 μm.

(D) 3D reconstruction corresponding to panel C. Left to right: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP^39TAG::ms2 in green in all panels). See also Video S1 for the animated 3D reconstructions. Scale bars: 10 μm.

(E) IF staining against PylRS and FISH staining of mRNA::boxB show recruitment of boxB-tagged mRNA into λ_N22-based OT organelles. Left to right: PylRS (magenta), mRNA::boxB (yellow), merged (NLS::EGFP^39TAG::ms2 in green). Scale bars: 20 μm.