BMC Cancer 2001, 1:19 http://www.biomedcentral.com/1471-2407/1/19 For analysis of phospho-STAT3, a different method was used to visualize the phosphorylated protein species. NRP-152 and NRP-154 cells were grown in the presence or absence of testosterone, as described above. Cells were fixed in Fix & Perm Medium A (Caltag) for 10 min at room temperature. After washing twice in PBS, cells were resuspended in ice-cold methanol with vortexing, then allow to sit for 15 min on ice. After washing twice in PBS, cells were resuspended in Fix & Perm Medium B (Caltag) and allowed to remain at room temperature for 30 min. Medium B contained 2 mg/ml goat Ig for block- ing non-specific binding. After washing three times (in- cluding a 30 min time in cold PBS for the first wash), the cells were incubated with rabbit anti-phospho-STAT3 (Biosource), 1 µ g Ab/106 cells in 100 µ l buffer. The Ab is specific for the phosphorylated form of STAT3; it does not bind to unphosphorylated STAT3 or to other phos- phorylated signaling intermediates. After incubating for 1 hr on ice, the cells were washed, with a long period in PBS for the first wash as described above. Next cells were incubated with phycoerythrin-labeled goat anti-rabbit F(ab2)' (Caltag) for 1 hr on ice, and washed as described. For analysis, cells were brought to 1 ml in PBS. All flow cytometric analyses were performed on a Becton-Dickin- son FACScan, using CellQuest software for acquisition and analysis. Treatment of NRP-152 and NRP-154 cells with dexameth- asone NRP-152 and NRP-154 cells or clones (see below) were seeded at 105 cells/well in microtiter plates in the pres- ence or absence of dexamethasone (Sigma) at 0.1 and 1 µ M. After 48 hr, NRP-152 and NRP-154 cells replicate wells of cells were harvested with either trypsin/EDTA (GIBCO) or 0.15 M NaCl/ 0.01 M Na citrate buffer (cit- rate-saline buffer), and the cells were processed for in- tracellular flow cytometry to analyze IL-6 production, as described above. Cloning NRP-154 cells by limit-dilution Washed NRP-154 cells were diluted to 10 cells/ml in complete medium, and 100 l/well of diluted cells were placed in wells of a microtiter plate. An additional 100 µ l/well complete medium were added, and the cells were incubated until growth was noted, 10 days later. At that time, 16/96 wells had cells growing in them (16.7% clon- ing efficiency), while the remaining wells did not. Medi- um was replaced, and plate was incubated until cells had grown enough to be removed to bigger wells. Clones were expanded, then analyzed for IL-6 production, as de- scribed above. Analysis of NRP-152 and NRP-154 cells for expression of IL-6 receptor Harvested NRP-152 and NRP-154 cells were washed twice in cold FACS buffer (PBS/0.1% serum/0.01% NaN3). Cells were blocked by incubation on ice in goat Ig (Sigma), 2 mg/ml, for 45 min. After washing twice, cells were incubated with 1 or 2 µ g/106 cells in 100 µ l bioti- nylated goat anti-human IL-6 receptor (ligand-affinity purified; R&D Systems) on ice for 45 min. After washing three times, cells were incubated with phycoerythrin-la- beled streptavidin for 45 min on ice. After washing three times, cells were analyzed on the flow cytometer. Treatment of NRP-152 and NRP-154 cells with AG490 The tyrphostin protein kinase inhibitor AG490 was pur- chased from Calbiochem. It was dissolved in DMSO, and stored at -20°C in single-use aliquots. NRP-152 and cloned NRP-154 cells were placed in 60 mm wells, and treated with AG490 for 48 hrs. The cells were removed with trypsin, and stained after washing with FITC-an- nexin V (5 µ l/106 cells; Caltag) for 15 min at room tem- perature. Apoptotic cells (cells staining with FITC- annexin V) were quantified by measuring green fluores- cence in FL1 on the flow cytometer. CellQuest software was used to acquire and analyze the data. STATView software was used to perform statistical analyses. Results Determination of the phosphorylation state of STAT3 in NRP-152 & NRP-154 cells We had very preliminary data, from a microarray exper- iment comparing the RNA of NRP-152 to NRP-154 cells, which indicated that STAT3 might be over-expressed on NRP-154 cells relative to NRP-152 cells (data not shown). Since STAT3 is active only when phosphorylat- ed, we decided to confirm these preliminary results by looking for phospho-STAT3 in both cell lines by intracel- lular flow cytometry. We observed that STAT3 was con- stitutively phosphorylated in NRP-154, but not NRP-152 cells. Even when treated with testosterone as described in Materials and Methods, NRP-152 cells did not exhibit phosphorylated STAT3 (Figure 1). These data indicate that the over-expressed STAT3 observed in NRP-154 cells in the gene microarray was putatively active as a transcription factor, since the amount of phospho- STAT3 in NRP-154 cells was increased relative to NRP- 152 cells. IL-6 is produced constitutively by NRP-152 and NRP-154 cells In order to determine if IL-6 was the activating ligand for phosphorylation of STAT3, we examined IL-6 expression in untreated NRP-152 and NRP-154 cells by intracellular flow cytometry. While both cell lines made IL-6, NRP- 154 cells displayed a different pattern, in that there was a