Figure 2.

IN core domain binding inhibitors reduce HIV IN nuclear accumulation. Nuclear import of DTAF‐labelled IN (DTAF‐IN) was reconstituted in mechanically perforated hepatoma tissue culture cells in the presence of exogenous cytosol and an ATP regeneration system as described in Section 4. (a) Confocal laser scanning microscopy (CLSM) images were acquired periodically for accumulation of DTAF‐IN (green panels) into intact nuclei in the absence or presence of the indicated inhibitors. Representative images for each sample are shown at 3 min and 20 min accumulation. Nuclear integrity was confirmed by the exclusion of a Texas Red‐labelled 70‐kDa dextran (TD70, red panels), in which intact cells appear as large black cells (denoted by white *), nuclei with intact nuclear envelopes appear as smaller TD70 excluding areas (marked by white arrows) and overperforated nuclei with damaged/leaky nuclear envelopes fail to exclude the TD70 properly (marked by white arrowheads). (b) CLSM images of accumulation of DTAF‐IN in the absence of an intact nuclear membrane (indicated by the lack of exclusion of TD70), induced by the addition of 0.025% CHAPS. (c) Image analysis was performed on the CLSM images such as those in (a, b) using ImageJ (NIH) software as described in Section 4. Each point represents the mean ± scanning electron microscope (n > 10). Note that in the absence of an intact nuclear membrane, steady state is reached within minutes; therefore, the data for the CHAPS‐treated samples are only presented for the first 10 min. Bud, budesonide; Mif, mifepristone; No Add, No addition