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. 2013 Aug 14;8(8):e72464. doi: 10.1371/journal.pone.0072464

Figure 1. EF24 inhibits radiation-induced DNA binding activity and transcriptional activation of NFκB; transactivation and secretion of TNFα in human neuroblastoma cells.

Figure 1

(A) NFκB DNA-binding activity in SH-5Y5Y and IMR-32 cells treated with EF24 (200nM) and exposed to 2Gy. The cells were incubated in a CO2/air incubator for additional 1, 3, 6, 24, 48 and 72h. The nuclear extracts were analyzed by EMSA using γ-32p[ATP] labeled NFκB-specific probe. Autorads were overexposed to capture the reduced activities that were equal or lower than mock-IR controls. (B) Luciferase reporter assay: SH-SY5Y, IMR-32, SK–PN–DW and MC-IXC cells transfected with pNFκB-Luc construct and either mock irradiated, exposed to 2Gy, or treated with EF24 and exposed to 2Gy were harvested at 24h post-IR and analyzed by luciferase assay. Data shown represent the mean and SD of three independent experiments. (C) Semi-quantitative densitometry of immunoblots using Quantity One 1D image analysis Version 4.6.5 (Biorad) showing α-tubulin intensity normalized expression of pIκBα in SH-SY5Y, IMR-32, SK–PN–DW and MC-IXC cells. Cells either mock-irradiated, exposed to 2Gy and harvested after 24h, treated with EF24 for 3h followed by 2Gy exposure and harvested after 1, 3, 12, 24, 48 and 72h, transfected with p50/p65 for 24h or transfected with p50/p65 for 24h and treated with EF24 for additional 24h. Groups were compared using Two-way ANOVA with Bonferroni’s Post-hoc correction. (D & E) Real time QPCR analysis showing TNFα mRNA expression in human neuroblastoma cells: (D) SH-SY5Y and (E) IMR-32 cells exposed to IR (2Gy) with or without EF24 treatment, transfected with RelA siRNA and exposed to 2Gy or treated with TNFR1 Ab and exposed to 2Gy and harvested after 1, 3 and 24h. The ΔΔct values were calculated by normalizing the gene expression levels to internal housekeeping gene (β-actin), compared between groups, and the relative expression level was expressed as a fold change over mock-IR cells. (F & G) ELISA analysis showing intercellular TNFα levels in (F) SH-SY5Y and (G) IMR-32 cells either exposed to IR (2Gy) with or without EF24, transfected with RelA siRNA and exposed to IR or treated with TNFR1 Ab and exposed to 2Gy. Conditioned medium from the cells were recovered after 24, 48 or 72h, concentrated (9KD concentrators) and subjected to ELISA. Group-wise comparisons were made using ANOVA with Tukey’s post-hoc correction.