Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 survival (Promega, Catalogue #G3580). Data elaboration of cell survival, IC50, and drug combination effects were performed according to previously described methods [11]. FACS Analysis Propidium iodide staining and FACS analysis were performed as described previously [9]. Annexin stain- ing was performed using Annexin V-FITCH Apoptosis Detection Kit I (BD Pharmingen Catalogue #556547) according to the manufacturer’s instructions. Data was elaborated using CellQuest Pro program (BD Pharmingen). Statistical analysis Data was analyzed using GraphPad Prism by applying the ANOVA test or t-test. Immunoblot analysis Immunoblot analysis was carried out as described pre- viously with minor modifications [9]. Dry milk was used as a blocking agent for the following antibodies: p53 (Santa Cruz Catalogue #sc-126), cyclin A (Santa Cruz Catalogue #sc-239), cyclin B1 (Santa Cruz Catalogue #sc-752), cyclin E (LabVision Catalogue #MS-870-P), cyclin D1 (Lab Vision Catalogue #RB-010-P), E2F-1 (Santa Cruz Catalogue #sc-251), GAPDH (Chemicon Catalogue #MAB374) and Rb (Santa Cruz Catalogue #sc-102); and bovine serum albumin for the following antibodies: cdk 2 (Santa Cruz Catalogue #sc-163), cdk 4 (Santa Cruz Catalogue #sc-260), cdk 6 (Santa Cruz Cat- alogue #sc-7961) and p21 (Calbiochem Catalogue #OP64). Chemiluminescence detection was achieved using SuperSignal West Pico Chemiluminescence Sub- strate (Pierce Catalogue #34080). RNA interference MON cells were plated at 300,000 cells/well in 6-well plates one day before transfection. Cells were transfected using DharmaFECT siRNA transfection reagent (Dhar- macon Catalogue #T-200(01-07)-01) according to the manufacturer’s instruction and using previously pub- lished p53 siRNA (Qiagen Catalogue #024849) and con- trol (Cy3-Luciferase GL2 Duplex, Dharmacon Catalogue #D-001110-01-05). One day post-transfection, cells were split into 50,000 cells/well using medium containing 10% charcoal and dextran treated fetal bovine serum, for drug treatment the following day. Immunocytochemistry MON cells were grown on glass coverslips and treated with drugs. Cells were fixed with 4% paraformaldehyde- PBS solution for 10 minutes, and permeabilized with 0.1% triton-PBS solution for 10 minutes. Cover-slips Page 3 of 13 were treated for 1 hour at room temperature with 1:250 diluted ap21 antibody (Calbiochem Catalogue #OP64). Staining was detected using the Vectastain ABC kit (Vector Laboratories Catalogue #PK-6102) according to manufacturer’s instruction. Peroxidase staining was developed using the DAB enhanced liquid substrate sys- tem (SIGMA Catalogue #D3939-1SET). The percentage of cells with nuclear expression of p21 was quantified by counting 250 to 300 individual cells, noting whether or not their nuclei showed positive staining above the background. The background staining was defined as an intensity of staining at or below the intensity of the negative control (i.e. any background staining that occurred in the absence of a p21-specific antibody). Caspase-profiling assays Caspase 3/7 Assay was performed using the Caspase-Glo 3/7 Assay kit (Promega Catalogue #G8093). Caspase activity profiling assays were performed using ApoAlert Caspase Profiling Plate (Clontech Catalogue #630225), according to the manufactures’ instruction with minor modification, using 50 μg of protein for each well of the assay. Results and Discussion Combination treatment with flavopiridol and 4OH-Tam inhibits the growth of RT cells To determine the effect of combining flavopiridol with 4OH-Tam, we treated MON, G401, and A204 RT cells with increasing concentrations of flavopiridol (0 to 200 nM) in combination with 2.5, 5.0, or 10.0 μM 4OH- Tam for two days. Flavopiridol inhibited RT cell growth with IC50 values of ~50-100 nM, with MON cells show- ing an intermediate level of sensitivity compared to G401 and A204 cells (Figure 1A-D). Exposure of all cells to flavopiridol and 4OH-Tam in combination resulted in a dramatic reduction in IC50 values, with 10 μM 4OH-Tam reducing the IC50s to ~0.12-0.2 nM (Fig- ure 1A-D). Furthermore, increasing the time of exposure to the combination of drugs enhanced the inhibitory effects in both MON and G401 cells (compare Figures 1A and 1B with Figure 2A, B, E, and 2F). Bar graphs derived from the data in Figure 2A and 2E are given to show the decreases in cell survival induced by the speci- fic concentrations of the drugs after three days of expo- sure (Figure 2D and 2H). We also tested the effect of 4OH-Tam alone and found that 4OH-Tam decreased cell survival significantly (Figure 2C and 2G). However, increasing time of exposure to 4OH-Tam alone from one to three or five days, did not result in further decreases of cell survival (Figure 2C and 2G). Thus, while the flavopiridol and 4OH-Tam combination resulted in a time dependent decrease of survival, the effect of 4OH-Tam alone was independent of time of