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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 S4 — Separate OT organelle systems co-localize MCP, PylRS, tRNA^Pyl, and ribosomes, related to Figure 3

(A) Overview cartoon of the constructs used. The lowercase “s” indicates that the PylRS and the MCP are expressed from two separate plasmids and not genetically fused.

(B–D) 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. Scale bars: 20 μm.

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

(C) IF staining against PylRS and RPL26L1 show that endogenous ribosomes co-localize with OT film-like organelles. Note that as RPL26L1 is an endogenous protein, also untransfected cells are stained. Top to bottom: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP^39TAG::ms2 in green).

(D) IF staining against PylRS and MCP (with an anti-HA antibody) show co-localization in OT organelles. Top to bottom: PylRS (magenta), MCP (yellow), merged (NLS::EGFP^39TAG::ms2 in green).

(E,F) HEK293T cells expressing the indicated OT organelle, tRNA^Pyl and a construct encoding EGFP^39TAG::boxB in the presence of SCO-K. All images show a selected plane of a z stack. Scale bars: 20 μm.

(E) IF staining against PylRS and λ_N22 (with an anti-Myc antibody) show co-localization in OT organelles. Left to right: PylRS (magenta), λ_N22 (yellow), merged (NLS::EGFP^39TAG::boxB in green).

(F) IF staining against PylRS and RPL26L1 show 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::boxB in green).