Contribution of Different Sites in KSHV LANA CTD to the Formation of LANA Nuclear ‘Speckles’.
A: Speckle formation assay with kLANA mutants of different sites. HeLa cells were transfected with vector containing 4xTR and kLANA wt or mutant vectors. kLANA was stained with a mouse αLANA antibody and DNA was stained with DAPI. Expression of GFP confirms the presence of the TR containing vector in the cells. Close up of an exemplary single cell from each of the images is shown in the top row. Images taken at 63× magnification, a scale bar of 20 µm is shown in the DAPI image of the first sample (wt+GFP). B: Quantification of the number of LANA speckles per nucleus performed with Cell Profiler software. The mean LANA speckle number per nucleus is plotted on the graph for each of the LANA proteins. A total of 80–110 cells were analyzed per sample in two independent experiments. The error bars indicate SEM. Speckle numbers in the samples marked in red are significantly different from those obtained with wt LANA.