Figure 2.
Super-resolution microscopy in combination with fluorophore counting provides a lower limit estimate of the copies of Nup107 per NPC. (A) Endogenous Nup107 was efficiently replaced with Nup107 N-terminally fused to mEos2. HEK293-Flp-In-T-REx cells were stably transfected with two microRNAs targeting both UTRs of the Nup107 gene (miRNAs-αNup107, lanes 1–4) or with a vector containing the same miRNA cassette together with the human mEos2-Nup107 fusion (mEos2-Nup107_miRNAs-αNup107, lanes 5–8), both under an inducible promoter. Nuclear extracts were analyzed by western blot and probed with antibodies against Nup107 (top panel) or GAPDH (bottom panel), as loading control. (B) Representative super-resolution image of a nucleus isolated from transfected HEK293 cells shows the characteristic circular NPC staining. Zoomed images of selected nuclear pores are shown together with the respective number of counted fluorophores (after correction for labeling efficiency; scale bars: 1 μm and 50 nm for zoomed images). (C) Distribution of the measured copies of mEos2-Nup107 per NPC obtained from super-resolution-based fluorophore counting (after correction for the labeling efficiency). The majority of all measured NPCs contain >16 copies of mEos2-Nup107. The final copy number frequency distribution was obtained from 172 clusters imaged from 5 nuclei and corrected for labeling efficiency. See also Supplementary Figures S3 and S4, and Supplementary Movie S1. Source data for this figure is available on the online supplementary information page.
