Figure 4

The detection and location of FNDs in the ECS cells.

(a) P19 cells were plated a density of at 7 × 10⁵ cells per 60-mm Petri dish for 16–20 h. Then the cells were treated with or without FNDs (50 μg/ml for 24 h). At the end of treatment, the cells were incubated with mouse anti-SSEA-1 antibody and then stained with goat anti-mouse IgG Cy3 antibody. The nuclei were stained with Hoechst 33258. The fluorescence intensity of FNDs was excited with wavelength 580 nm and the emission was collected in 600-700 nm. The fluorescence intensity of nuclei was excited with 405 nm and the emission was collected in 415–485 nm. The fluorescence intensity of SSEA-1 was excited with 543 nm and the emission was collected in 560–580 nm. The nuclei display blue color. SSEA-1 displays yellow color. FND displays red fluorescence. The arrows indicate the location of FND in cytoplasm. (b) P19 cells were plated at a density of 7 × 10⁵ cells per 60-mm Petri dish for 16–20 h. Then the cells were treated with or without FNDs (50 μg/ml for 24 h). At the end of treatment, the total proteins of cell extracts were collected and subjected to Western blot analysis using specific anti-SSEA-1 and anti-actin antibodies. Actin was a loading control. The representative Western blot data were shown from one of three separate experiments with similar findings.