Figure 2

InlP translocates to the nucleus and changes the shape of splicing speckles. (A) Left, representative immunofluorescence images of HeLa cells transfected with InlP-_HA or Lmo2027-_HA and stained with an HA antibody and DAPI.  Right, corresponding pixel intensity plot for white lines drawn on the left. Scale bar 10 µm. (B) Nucleus to cytoplasm fluorescence intensity ratios of InlP-_HA or Lmo2027-_HA, as determined with ICY software. Data are mean ± SD of triplicate experiments (n = 22 cells per replicate). (C) Immunofluorescence of InlP-_HA or Lmo2027-_HA and endogenous SC35 in HeLa cells transfected as in (A) and stained with HA and SC35 antibodies and Hoechst. Scale bar 10 µm. Representative SC35-positive speckles are enlarged in squares 1 and 4 for non-transfected cells, in square 2 for InlP-transfected cells, and in square 3 for Lmo2027-transfected cells. (D) Nuclear speckle circularity in HeLa cells transfected with InlP-_HA. On the top is a representative image stained as in (C) with SC35-positive nuclear speckles in red. Below, the color-coded representation of nuclear speckle circularity index. (E) Scatter plot of the circularity index of nuclear speckles from at least 20 cells transfected as in (C,D) determined by ImageJ software. The bar represents the median. Statistical significance determined by ANOVA.