Fig. 5

HAS1 expression induces centrosomal abnormalities. a MCF10A cells transfected with indicated plasmids were selected followed by centrosome staining using an antibody against pericentrin. The nucleus was stained with DAPI (blue). The left column shows the composite image of pericentrin (white) and nucleus (blue). The red and green scale bars on the left-panels are 10 μm each. The right-top-panels show zoom-in of the indicated area from the left panels (pericentrin as green). Bottom right panels are the magnification of the indicated dotted area from the right-top panels to visualize only pericentrin staining (green). The white bar is 1 μm in lower right panels. b Representative images of centrosomal abnormalities in DLD1 cells (left panel) and HeLa cells (right panel). Cells expressing HAS1 (middle panels) for both cell lines show centrosomal abnormalities but no such event was observed in pTET (upper panels) and HAS2 (lower panels) expressing cells. The cells population were immunostained for pericentrin (red) and the nucleus stained with DAPI (blue). Tetracycline-inducible lines (DLD1 and HeLa) were passaged though continuous Dox exposure to induce HAS1 and HAS2 mediated HA synthesis for many generations (10 weeks). The pTET cells were used as controls. c The average fluorescence intensity of pericentrin staining per nucleus was calculated from at least four random regions of interest (ROI) and represented with the standard errors. The DLD1 cells expressing HAS1 has higher intensity than DLD1 cells alone, pTET and HAS2 expressing cells. Relative fluorescent intensity of the red channel (pericentrin) from the confocal images was collected for ROI and the total number of nucleus was counted. Multiple focused- or Z-stacking images were used to compile these composite images for complete coverage across the thickness of the cells