PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery
Fig 2
PML protein encompasses incoming viral genomes only after nuclear delivery.
HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized, and treated with Click-iT reaction buffer with AF555 dye to stain EdU-labeled pseudogenomes (red). Next, cells were incubated with mouse anti-PML protein antibody (green) and mounted in DAPI (white). (A) Images show 3D reconstruction of high resolution z-stacks and close-up images were rotated at a 45° angle on the x, y, z axes in the 3D-rendered images. (B) Distance (μm) between center of EdU and center of PML protein puncta was measured in NIS Elements. (C) PML and EdU mean intensity were measured in NIS Elements, normalized to ROI area, and the ratio was calculated. (B and C) Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. Results are shown as average of 2 independent experiments and SEM, with 30–50 cells of each condition (early and late interphase cells) collected in z-stacks spanning the whole nucleus. (B) 7+ nucleoli: 0.275 μm ± 0.021 μm; 1–6 nucleoli: 0.113 μm ± 0.006 μm. (C) 7+ nucleoli: 1.146 ± 0.025; 1–6 nucleoli: 1.888 ± 0.069. p value was determined using Student’s t-test comparing Early to Late Interphase. ****: p < 0.0001.