Fig. 5: UV-induced blocking of NPCs by DAPI/ Hoechst.
From: Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide
HeLa cells were grown in cell culture wells, permeabilized with digitonin, and preincubated without or with the traditional DNA markers DAPI or Hoechst 33342. Indicated wells were then partially exposed for 30 s with UV light from the microscope’s mercury/metal halide lamp (wavelength ≈ 365 nm). A mixture of importin β, IBB-sinGFP4a (an importin β-dependent import cargo), mCherry (as a passive exclusion marker), components of the Ran system, and an ATP/GTP-regenerating system (“Methods”) was added. Import, followed live by confocal laser-scanning microscopy, was allowed to proceed at 21 °C. The micrographs show the ≈200 s time point of either a non-UV exposed area or a fully exposed one (“full-field”). “Half-field” indicates the region where the illumination boundary (dashed line) had crossed. Strong accumulation of IBB-sinGFP4a was observed in nuclei not pre-exposed to UV or samples without DAPI/Hoechst addition. The combination of DAPI or Hoechst with UV illumination blocked the import of IBB-sinGFP4a completely.
