Fig. 3

RNAi knockdown of ST8SIA2 decreases expression of membrane-associated dPSA and nucleolin but not nuclear nucleolin. RNAi was used to knockdown expression of ST8SIA2 in SK-MEL-28 human melanoma cells and the effect on dPSA and nucleolin production was determined by confocal laser scanning microscopy. A The absolute ST8SIA2 and ST8SIA4 mRNA copy number relative to that of GAPDH in parent SK-MEL-28 cells. N = 3. B Relative expression of mRNA coding for ST8SIA2 and ST8SIA4 polysialyltransferases in parent, a scrambled RNA negative control and ST8SIA2 RNAi knockdown SK-MEL-28 mutants measured by RT-PCR. N = 3. Brightfield micrographs of (C) scrambled control compared to (D) ST8SIA2 knockdown mutant show morphological differences. Confocal laser scanning micrographs of the scrambled and ST8SIA2 knockdown mutant show dPSA and nucleolin are co-localized on (E) the surface and (F) inside cells but are both decreased when ST8SIA2 is knocked down except for nucleolin in the nuclear membrane. dPSA and nucleolin were identified on the surface (E–F, upper panels) or inside (treated with Triton X-100; E–F, lower panels) SK-MEl-28 cells with anti-dPSA mAb SEAM 2 or anti-nucleolin mAb MS-3, respectively, and detected by fluorescently labeling with Alexa Fluor 594 (red fluorescence) or Alexa Fluor 488 (green fluorescence)-conjugated anti-mouse subtype-specific antibodies using confocal laser scanning microscopy. Blue fluorescence is DAPI DNA staining. Scale bars, 20 μm