Figure 2

Peptide uptake by prostate cancer cell lines. Figure 2A: Enhancement of Oct6 NLS peptide uptake by addition of Glu-Ala peptide EEEAA on the N terminus. DU-145 cells were incubated with 500 nM peptides for 4 hours in Materials and Methods. Harvested washed cells were subjected to flow cytometry on a FACScan. Dead cells were excluded by gating on 7-AAD-negative cells. * = geometric MFI of Glu-Lys-FITC or Glu-Oct6-FITC was significantly greater than Glu-FITC (p < 0.01) or 356 (p < 0.05) by paired ANOVA. The average of 3 replicate experiments is shown. Figure 2B: Effect of concentration on uptake of Glu-Oct6-FITC by DU-145, LNCaP and PANC-1 cells. Concentrations of Glu-Oct6-FITC ranging from 0 to 300 nM were incubated with cells for 1 hr, at which time cells were harvested, washed, and processed for flow cytometry. Figure 2C: Effect of substituting Phe, Asn, or Tyr for Glu on Oct6 NLS peptide uptake by DU-145 and PANC-1 cells. Cells were incubated with 0 to 1000 nM of peptides for 1 hr at 37°C, then were harvested, washed, counterstained, and analyzed by flow cytometry to quantify fluorescence. The average of 3 independent experiments ± SD is shown. Figure 2D: Lack of competition by Phe-Oct6-Dansyl or Asn-Oct6-Dansyl on Glu-Oct6-FITC uptake by DU-145 cells. 300 nM of either Phe-Oct6-Dansyl or Asn-Oct6-Dansyl were incubated with 300 nM Glu-Oct6-FITC for 1 hr, at which time cells were harvested, washed, and analyzed on a BD LSR II for dansyl and FITC fluorescence. CellQuest Pro was used to analyze the fluorescence data. Turquoise line indicates dansyl fluorescence intensity; green line indicates fluorescein fluorescence intensity. Figure 2E: Effect of 250-1000 nM Phe-Oct6 on 500 nM Glu-Oct6-FITC. DU-145 cells were incubated with peptides as before for 4 hrs; cells were harvested, washed, fixed, then analyzed for fluorescence by flow cytometry. Green = no peptide; pink = Glu-Oct6-FITC alone; blue = Glu-Oct6-FITC + 250 nM Phe-Oct6; red = Glu-Oct6-FITC + 500 nM Phe-Oct6; purple = Glu-Oct6-FITC + 1000 nM Phe-Oct6.