the respective protein expression levels between the EKB treated, IR exposed and control groups. Blots were stripped and reprobed with mouse monoclonal anti-a-tubulin antibody (Santa Cruz) to determine equal loading of the samples. One diamentional gel analysis was performed using a BioRad Multi-Analyst software package with an integrated density program. Group-wise com- parisons were made using ANOVA with Tukey’s post-hoc correction. A P value of ,0.05 is considered as statistically significant. Real-Time QPCR profiling of NFkB signaling pathway molecules Total RNA extraction and real-time QPCR profiling were performed as described in our earlier studies [29] . We used human NFkB signaling pathway profiler (Realtimeprimers.com, Elkins Park, PA) containing 88 genes representing 8 functional groups including (i) Rel/NFkB/IkB family, (ii) NFkB responsive genes, (iii) Ligands & Transmembrane receptors, (iv) Adaptor proteins, (v) Signal transduction kinases, (vi) Transcription factors, (vii) Cell death/survival molecules, and (viii) Other factors. We started with this highly selected QPCR profiler instead of an all-encompassing gene array because the selected genes entail a well-characterized profile governing NFkB signal transduction and transcriptional targets, hence facilitating interpretation of data, simplifying data acquisition and analysis, and avoiding genes not functionally characterized. Furthermore, QPCR profiling allows detection and quantification of gene expression in real-time. Each profiling plate was also equipped with reverse transcription control, positive PCR control, genomic DNA control and five housekeeping genes – b- Actin, GAPDH, Rpl13a, HPRT1 and b2M. The DDct values were calculated by normalizing the gene expression levels to the expression of the housekeeping genes. The normalized data were then compared between groups, and the relative expression level of each gene was expressed as fold change. When comparing each gene’s signal intensity between groups, we used a twofold or more ($2 fold) increase or decrease to represent ‘‘stringent’’ criteria for upregulation or downregulation and an increase/decrease of ,2 fold to represent ‘‘less stringent’’ criteria. Classifying gene regulation criteria in this manner can provide an index of reliability of the gene expression data [29]. Cell Viability Trypan blue dye exclusion assay was used to identify IR modulated cell viability in HNSCC cells and further, to determine the efficacy of EKB-569 in this setting. Cells exposed to IR alone and cells pre-treated with EKB-569 followed by exposure to IR, were sequentially analyzed with the Countess automated cell counter (Carlsbad, CA). Furthermore, to determine the efficiency of EKB-569 in targeting IR-induced NFkB dependent cell viability, trypan blue exclusion assay was performed in NFkB over-expressed HNSCC cells exposed to EKB-569. Group-wise comparisons were made using ANOVA with Tukey’s post-hoc correction. A P value of ,0.05 is considered statistically significant. Cell survival by MTT assay Cell survival was analyzed using MTT assay as described in our previous studies [29]. HNSCC cells at a density of 1000 cells/ 300 ml in a 24-well plate were either (i) mock-irradiated, (ii) exposed to IR alone, (iii) treated with EKB-569 (0.5, 1.0, 2.0 and 5.0 mg) alone, (iv) pretreated with EKB-569 (5.0 mg) followed by exposure to IR, (v) prior transfection with DIkBa followed by exposure to IR, or (vi) prior transfection with p50/p65 treated EKB Radiosensitizes Squamous Cell Carcinoma with or without EKB-569. The treated and/or exposed cells were added with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoli- um bromide (30 mL/well from 5 mg/mL stock) for 4 h after 24, 48 and 72 h of post-IR. Solubilization of converted purple formazan dye was accomplished by acid-isopropanol with continuous shaking at 37uC. The reaction product was quantified by measuring the absorbance at 570 nm using Synergy II micro plate reader (Biotek). Cell survival response was compared using ANOVA with Tukey’s post-hoc correction. Nuclear morphology by dual staining SCC-4 cells (56105 cells in 500 ml of complete growth medium) grown in 4-well plate (Nunc) were either: 1) sham treated, 2) treated with EKB-569 (0.5–5.0 mg), 3) exposed to IR with or without prior EKB-569/DIkBa transfection, or 4) transfected with p50/p65 subunit with or without prior EKB-569 treatment. The cells were analyzed for nuclear morphology as described earlier [30]. In brief, the medium was replaced with a fresh medium containing reduced serum (2%) without any added growth factors and incubated further for 16 h at 37uC in air/CO2 incubator. The cells were then stained with acridine orange (1 mg/ml) and ethidium bromide (1 mg/ml) and immediately examined for the morphological characteristics of apoptosis at 2006 magnification using an Olympus VANOX fluorescent microscope. Four morphological states were examined: (1) viable cells with normal nuclei (bright green chromatin with organized structure); (2) viable cells with apoptotic nuclei (green chromatin which are highly condensed and/or fragmented); (3) non-viable cells with normal nuclei (bright orange chromatin with organized structure); and (4) non-viable cells with apoptotic nuclei (bright orange chromatin which is highly condensed or fragmented). Results EKB-569 selectively inhibits IR-induced persistent activation of NFkB The effect of EKB-569 in selectively inhibiting IR-induced NFkB-DNA binding activity was elucidated using four different approaches. First, we investigated whether EKB-569 as a stand- alone compound, could modulate NFkB activity in both SCC-4 and SCC-9 HNSCC cells. Compared to untreated cells, EKB-569 treatment dose-dependently inhibited NFkB DNA binding activity with a substantial inhibition at 5.0 mg (Fig. 1A & B). Next, to unveil the radiosensitizing efficacy of EKB-569, HNSCC cells mock-irradiated, exposed to IR or treated with EKB-569 (0.5, 1.0, 2.0 or 5.0 mg) and then exposed to IR were analyzed for alterations in NFkB activity. Unlike the mock-IR controls, IR at 2 Gy significantly (P,0.001) induced NFkB-DNA binding activity in both SCC-4 and SCC-9 cells (Figure 1 A & B, bottom panel). This IR-induced NFkB activity was drastically (P,0.001) inhibited with EKB-569 treatment in a dose dependent manner (Fig. 1D) in both cell types. It is interesting to note that at 5.0 mg concentration, EKB-569 completely suppressed IR-induced NFkB activity even below the constitutive (mock-IR) levels in this setting. Further, to delineate whether EKB-569 persistently inhibits IR- induced NFkB or there is recovery of IR-induced NF-kB activity over time, SCC-4 cells pretreated with EKB-569 and exposed to IR were examined for 3 days post-radiation exposure. EKB-569- induced inhibition of IR-induced NFkB DNA-binding activity remained at the same decreased level at all time points investigated (Figure 1E). Densitometric analysis revealed a significant (P,0.001) inhibition of IR-induced NFkB DNA-binding activity up to at least 3 days post-radiation exposure. (Fig. 1F). To confirm the specificity of the EMSA band seen in Figure 1 A and B, a PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29705