Figure 2

<p>Cytotoxic effect of 5-FU on non-sorted and sorted (uPAR-positive and uPAR-negative populations) derived from SCLC cell lines. (A) 1×10⁴ cells (H211, H69AR, H1417) were placed in wells of a 48-well plate in triplicates and incubated for 72 hr in the presence of varying concentrations of 5-FU. (B) SCLC cell lines were FACS sorted after staining with anti-uPAR antibodies and were plated at the same seeding density (4×10³/well of 96-well plate) and treated with 5-FU at 0, 10, 100, 200 µg/ml for 72 hr. Cell survival was evaluated after adding Guava ViaCount reagent and counting viable and dead cells. Only viable cells were included in data analysis, and 100% viability was defined as number of viable cells cultured in absence of 5-FU. Statistical analysis (2-way ANOVA) of uPAR(+) and uPAR(−) data sets revealed significant differences among viability of uPAR(+) and uPAR(−) cells (<i>P</i> = 0.0002, 0.0027, 0.0008 for H211, H69AR, H1417 cells, respectively). The data points represent averages±SD of three independent experiments.</p

Figure 3

<p>Cytotoxic effect of cisplatin and etoposide on non-sorted cells derived from SCLC cell lines. (A) SCLC cell lines (non-sorted) treated with cisplatin, etoposide at concentrations 0, 3, 10, 100 µg/ml or their combinations (cisplatin and etoposide at final concentrations of 10 µg/ml, 100 µg/ml) for 72 hr. Cell survival was evaluated after addition of Guava ViaCount reagent and counting of both surviving and dead cells using Guava ViaCount software. Data were normalized as 100% viability of cells cultured in absence of drugs. Error bars indicate standard deviation of triplicate cultures (results of three independent experiments). (B) After treatment cisplatin and etoposide, viable adherent cells were detached by trypsin treatment and were stained with anti-uPAR-FITC antibodies and percentage of uPAR-positive cells was determined by FACS analysis. Sample with mouse IgG isotype control antibody was used to set the value of the FACS gate, which was applied to all samples stained with uPAR-FITC.</p

Figure 4

<p>Colony-forming activity of uPAR-positive and uPAR-negative cells derived from SCLC cell lines. (A) H1417- derived, uPAR-positive sorted cells formed multiple colonies in methylcellulose media, while uPAR-negative cells from the same sorts displayed little or no clonogenic activity. (B) Graphical representation of colony-forming ability of uPAR-positive and uPAR-negative cells at different plating densities 3000, 1000, 100 cells/6-well plate (H1417, H69AR, H211). (C) Distribution of uPAR-positive cells in the colonies derived from sorted uPAR-positive cells grown in methylcellulose media. A total of 20 cell colonies from the H1417 cell line were analyzed.</p

Figure 5

<p>Expression of CD44 and MDR1 on uPAR-positive and uPAR-negative cells. (A) FACS analysis of, H211, H69AR and H1417 SCLC cell lines double-labeled with uPAR-FITC and CD44-PE, MDR1-PE. The percentages of cells expressing CD44 and MDR1 were calculated separately for uPAR-positive and uPAR-negative cells. (B) Fluorescent microscopic analysis of double-labeled and FACS-sorted cells. Examples of uPAR-FITC/CD44-PE double-labeling (a,b,c) and uPAR-FITC/MDR1-PE double-labeling (d,e,f). (Bf-inset) H1417 cell line stained with mouse IgG isotype control-PE (red), isotype control-FITC (green) and DAPI (blue).</p