Fig 2: GSK3β inhibition induced TPR mislocalization, leading to its proteasomal degradation(A) HCT116 cells were treated with DMSO (control), AR-A014418 (AR), or SB-216763 (SB), and then synchronized at mitotic phase as shown in Figure 2E. Immunoprecipitates (IP) from extracts of mitotic HCT116 cells with anti-GSK3β (left panels) and anti-TPR (right panels) antibodies, and nonspecific rabbit IgG, were analyzed by immunoblotting with indicated antibodies (arrowheads indicate TPR and its isoform). (B) qRT-PCR analysis of TPR, DYNEIN, and NUP153 mRNA in HCT116 cells after treatment with DMSO or GSK3β inhibitors (AR-A014418 and SB-216763) for 48 hours. Data show mean ± SD of three separate experiments. (C) Expression of TPR in HCT116 cells treated with DMSO (C), 25 μM AR-A014418 (AR), or SB-216763 (SB) for 72 hours in the presence or absence of MG132 (10 μM) for 8 hours.

Fig 3: Short-range association between integration sites and genomic features.(a, b) Histograms show the distribution of absolute genomic distances to specific features of HIV-1 integration sites, with bin size of 200. The genomic features are Histone methylation H3K36me3 and H3K4me3 in a 2 kb window centred on integration sites. The distributions are shown for control cells (orange bars), Tpr KD cells (light blue bars) and as predicted for random integration (grey bars). (c) Frequency of ISs from histone methylation H3K36me3 and (d) H3K4me3, as function of genomic distance, plotted with bins of 100 bp in a ±50 kb window centred on integration sites in control cells (left, orange) and Tpr KD cells (right, blue). Dashed curves correspond to kernel density estimations of the distance distribution for control cells (red), Tpr KD (blue) and random integration (grey).

Fig 4: Tpr is specifically involved in HIV-1 infection.(a) Three out of ~10 final clones obtained by limiting dilution were selected for the expression of Tpr by western blotting and (b) MLV-Luc and HIV-1-Luc were used in parallel to infect the control clone (obtained with an empty LV) and clones 2, 5 and 6. (c) Tat was transfected in all clones to evaluate LacZ expression by ß-galactosidase assay normalized to the amount of protein. Clones were thawed and frozen multiple times and yielded similar results (s.d. are shown). (d–f) Transcriptome profiles comparison: scatter plots show RPKM values observed in ~36,000 annotated in control sample (x axis) and Tpr KD (y axis). Scatter plots of Pearson correlation calculated using the RNA-seq log2 RPKM values of Tpr depleted versus control for (d) HeLa cells, (e) Jurkat cells and (f) clones.

Fig 5: Super-resolution imaging of active chromatin underneath NPCs in control and Tpr depleted HeLa cells.(a) STORM image (visualized as smoothed histogram of computed positions) of Nup153-A568 (green) and H3K36me3-Cy5 (red) in control cells (left) and Tpr KD cells (right). The top left corner of the figure shows the low resolution image that would have been obtained with a conventional (diffraction limited) microscope to appreciate the gain in resolution. The yellow spots outside of the nuclei are multicolour fluorescent beads used to correct sample drift and chromatic shifts. (b) STORM image (visualized as scatter plots of computed positions): zoom on a part of left image in panel a showing rectangles oriented across the NE and passing either through NPCs (solid boxes) or in between neighbouring NPCs (dashed boxes). The positions of boxes are defined manually based on the green (NPC or NE) channel only. These boxes are used to quantify the distribution of H3K36me3 in panel c. (c) Density profiles of H3K36me3 as function of distance from the NE, computed from the rectangular boxes as shown in b. Solid curves correspond to boxes passing through NPCs, dashed curves to boxes passing in between neighbouring NPCs. Black curves correspond to control cells, red curves to Tpr KD HeLa P4CCR5 cells. The profiles were computed as described in Methods from 122 NPCs and 305 positions between NPCs in Tpr KD cells versus 140 NPCs and 265 positions between NPCs in control cells; au, arbitrary units.