Interaction of Bcl-3 and CTCF gene resulted in suppressing CTCF expression in corneal epithelial cells. As shown above that UV stress-induced Bcl-3 activation downregulated CTCF, we further verified whether altered Bcl-3 activity has a Wortmannin functional impact on regulation of CTCF in UV stressinduced HCE cells. Bcl-3 was knocked down by transfecting cells with siRNA specific to Bcl-3, and control cells were transfected with non-related siRNA with/without UV irradiation. The results showed that knockdown of Bcl-3 resulted in increases in CTCF mRNA levels following a time course detected by RTPCR and by quantitative real-time PCR. In addition, knocking down Bcl-3 mRNA abolished the inhibitory effect of UV irradiation on CTCF promoter activity. The effects of UV irradiation on suppression of CTCF were reversed by silencing Bcl-3 mRNA indicate that Bcl-3 plays an important role in regulating CTCF transcription by control of CTCF promoter activity. Interaction between Bcl-3 and p50. In order to demonstrate the physical interaction between Bcl-3 and p50, Bcl-3 and p50 were pulled down each other by antibodies against Bcl-3 and p50 in EGF- and UV stress-induce cells, respectively. Bcl-3 antibody pulled down more p50 UV stress-induced HCE cell compared with cells stimulated with EGF. No bands were found in the lanes of IgG controls indicating that there was truly a difference of the p50 levels in UV and EGF stimulated cells. In the meantime, Bcl-3 was specifically immuno-coprecipitated with p50 by anti-p50 antibody in UV stress-induced cells. However, Bcl-3 did not interact with p50 in EGF-stimulated cells. Total amounts of p50 and Bcl-3 in cell lysates were analyzed by Western blots for input controls. To further investigate whether there is a functional interaction between Bcl-3 and p50, experiments to co-localize Bcl-3 and p50 in nuclei in EGF and UV stress-induced cells were performed using antibodies labeled with Alexa 488 and 594 against Bcl-3 and p50, respectively. Bcl-3 and p50 were co-localized in the nucleus that was indicated by DAPI staining in UV stress-induced cells. There was no co-localization of Bcl-3 and p50 found in EGFinduced and un-stimulated cells. Further statistical analysis revealed that there was a significant increase in Bcl-3 and p50 co-localization in nuclei of UV stress-induced cells. Immunoco-localization of Bcl-3 and p50 in the nucleus of UV stress-induced cells suggests that Bcl-3 and p50 are very likely to form a functional heterodimer complex in response to UV stress stimulation. The results provide further evidence indicating that Bcl-3 has functional interactions and physical associations with p50 in the nucleus of UV stress-stimulated HCE cells. In recent studies, we demonstrate that regulation of CTCF is stimulus-dependent to affect HCE cell fate. Differential stimuli involve activation of NF-kB subtypes that are able to regulate CTCF activity in the NF-kB family. However, there is no detailed information available regarding why UV stress-induced regulation of CTCF is different from the EGF effect since both stimuli all activate the NF-kB pathway.