Figure 4

Imaging studies. Figure 4A: DU-145 and LNCaP cells were incubated with 250 nM Glu-Ala-FITC or Glu-Oct6-FITC for 4 hours, counterstained with 5 mM DRAQ 5, then fixed in paraformaldehyde and analyzed by imaging flow cytometry on an Amnis Imaging Cytometer. IDEAS software (Amnis) was used to generate and colorize the images. Across each row of images, one is viewing the same cell under different fluorescence excitations. Thus for each cell type and peptide, the left-most column shows the merged images, the middle column shows the DRAQ5 images, and the right-most column shows the FITC images (labeled below each set of 3 channels as m for merge, D for DRAQ5, and F for FITC). Analysis using IDEAS software revealed that Glu-Oct6-FITC distributed about 50% in nuclei and 50% in cytoplasm of cells, whereas only ~10% of nuclei were positive for DRAQ5 and FITC colocalization in the case of Glu-Ala-FITC. Figure 4B: Live cell imaging of Glu-Oct6-FITC by DU-145 cells. 500 nM Glu-Oct6-FITC was incubated with DU-145 cells overnight (necessary for imaging purposes). Cells were washed with phenol red-free buffer, counter-stained with DRAQ5 at 10 mM, and then examined under a Zeiss Axiovert 200 inverted phase contrast microscope with epifluorescence. The merged image confirms the presence of Glu-Oct6-FITC in nuclei. Merged images with and without Nomarski interference are shown. Figure 4C and D: DU-145 cells incubated with Glu-FITC and Oct6-FITC, as described for Figure 3B. Note the absence of FITC fluorescence in nuclei. Figure 3D merged image shows the Nomarski interference image overlayed on the fluorescence images.