EKB Radiosensitizes Squamous Cell Carcinoma Figure 3. Real time QPCR profiling: Histograms showing IR-induced NFkB-dependent downstream signal transduction molecules and the effect of EKB-569 (5.0 mg) on these IR-modulated genes in human SCC-4 cells. doi:10.1371/journal.pone.0029705.g003 and analyzed for cell viability, survival and death. EMSA analysis (Figure 5A) confirmed the robust NFkB DNA-binding activity in p50/p65 transfected SCC-4 cells (Figure 5B). Further, over expression of NFkB in these cells significantly (P,0.001) induced cell survival (Figure 5C) and showed bright green chromatin with organized nuclear morphology (Figure 5E) and, served as positive controls for our study. Consequently, treatment with EKB-569 significantly (P,0.001) inhibited cell viability, survival and showed bright orange chromatin with blebbing, nuclear condensation, and fragmentation in these NFkB over-expressed cells (Figure 5C–E) Figure 4. Effect of EKB-569 on radiation modulated prosurvival signaling molecules, cell viability, survival and/or death. (A) Representative immunoblot showing expression levels of IkBa, pro-apoptotic Bax and anti-apoptotic Birc1, 2 and 5 in human SCC-4 cells exposed to IR or treated with EKB-569 (5.0 mg) prior to IR exposure. a-tubulin was used to show equal loading of protein samples. (B) Semi-quantitative 1D gel analysis showing increased IkBa and Bax levels in EKB-569 treated cells. EKB-569 treatment significantly suppressed Birc1, 2 and 5 in these IR-exposed SCC-4 cells. (C) Histograms showing the percent cell viability in cells treated with EKB-569 (0.5, 1.0, 2.0 and 5.0 mg). EKB-569 inflicted a dose dependent inhibition of cell viability in this setting. (D) Histograms showing the percent cell viability in cells either mock-irradiated, exposed to IRexposure or treated with EKB-569 (5.0 mg) and exposed to IR. Compared to the mock-IR cells, IR resulted in reduced cell viability. Relatively, EKB-569 treatment significantly conferred IR-inhibited cell viability. Cell viability was measured using Trypan-blue dye exclusion assay and counted in automated countess cell counter. (E) Cell survival in mock-IR, EKB-569 (0.5, 1.0, 2.0 and 5.0 mg) treated and in irradiated cells with or without EKB-569 treatment. MTT assay was used to analyze the induced cytotoxicity and the reaction product was quantified by measuring the absorbance at 570 nm. Percent cell survival was calculated as (mean of test wells/mean of control wells) 6100 and compared using ANOVA. EKB-569 induced a dose dependent inhibition of cell survival. Like-wise IR suppressed cell survival and this IR-inhibited cell survival was further inhibited with EKB-569 in a dose dependent fashion. (F) Nuclear morphology with dual staining showing apoptotic characteristics in cells either mock-IR, treated with EKB-569 (0.5, 1.0, 2.0, 5.0 mg), exposed to IR, or treated with EKB-569 and exposed to IR. Insert: High magnification photomicrographs showing chromatin with organized structures indicating viable cells with normal nuclei in untreated control cells and, chromatin with blebbing, nuclear condensation, and fragmentation indicating typical apoptotic characteristics in cells treated with 5.0 mg of EKB-569 and exposed to IR. doi:10.1371/journal.pone.0029705.g004 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29705