Figure S1.

Validation of Single Molecule Translation Visualization Assay, Related to Figure 1

(A–C) U2OS cells expressing scFv-GFP, PP7-2xmCherry-CAAX and the SunTag_24x-Kif18b-PP7_24x translation reporter. (A) Fluorescence intensity of mRNA foci was measured and was corrected for background fluorescence. The average corrected mRNA fluorescence intensity was set to 1 for each separate cell (n = 3 experiments, 14 cells, 278 mRNAs). (B) Intensity of single mRNA foci was measured and corrected for background, but intensity was not normalized as in (A) to allow comparison of absolute intensities (Black bars, n = 3 independent experiments, 22 cells, 377 mRNAs). In parallel, U2OS cells co-expressing SunTag_24x–CAAX and scFv-mCherry were imaged and the intensities of single membrane bound scFv-mCherry-SunTag_24x foci was measured (Red bars, n = 4 independent experiments, 24 cells, 162 mRNAs). (C) Dwell time of tethered mRNAs on the membrane. The time between mRNA appearance at the focal plane of the membrane and its disappearance was scored. mRNA disappearance was due to mRNA detachment or degradation, not photobleaching. Mean and SD are indicated.

(D) Cells expressing scFv-GFP with (left two images) or without (right two images) the SunTag_24x-Kif18b-PP7_24x reporter were imaged with indicated exposure time. Dotted line shows outline of the cell.

(E) U2OS cells expressing scFv-GFP, PP7-2xmCherry-CAAX and the BFP-Kif18b-PP7_24x translation reporter. Representative image is shown. Asterisk indicates lysosome.

Scale bars are 5 μm (D) and 2 μm (E).