Figure 4

Nuclear IN reorganization in the PIC occurs in the absence of LEDGF/p75-mediated chromatin tethering.

(A) Western blot showing LEDGF/p75 depletion in LEDGF/p75^KD cell lines and the complementation with the respective LEDGF/p75 truncations. (B) Representative confocal images of LEDGF/p75-depleted cells complemented with wild-type (LEDGF/p75^BC) or mutant LEDGF/p75^93–530 or LEDGF/p75^AT- during interphase and mitosis, stained for DAPI (blue) and immunostained for LEDGF/p75 (red). LEDGF/p75^BC but not LEDGF/p75^93–530 binds mitotic chromatin. (C) Mean FRET ratios of VSV-G pseudotyped viral complexes (HIV_{IN-mTFP1+IN-mVenus}) in the stable cell lines expressing the LEDGF/p75 constructs (LEDGF/p75^93–530, LEDGF/p75^AT- and LEDGF/p75^BC) compared to LEDGF/p75^KD cells. (D) Mean FRET ratios of VSV-G pseudotyped viral complexes (HIV_{IN-mTFP1+IN-mVenus}) in LEDGF/p75^325–530 and LEDGF/p75^{325–530;D366N} transfected LEDGF/p75^KD cells. White bars: cytoplasm; grey bars: nucleus; KD = knockdown; BC = back-complemented. Unless explicitly stated otherwise, the intensity and FRET ratios in the cytoplasm and nucleus were statistically different (p-value < 0.001). (***p-value < 0.001, *p-value < 0.05 and n.s. = not significant).