Cell-based Screening and Validation of Human Novel Gene TMEM9B Activated Nuclear Factor Signaling Pathway / 生物化学与生物物理进展

Nuclear factor κB (NF-κB) is an important cellular transcription factor. The important role of NF-κB-mediated cell signal transduction pathway in apoptosis is a hot topic at home and abroad. In order to discover new regulators in NF-κB signaling pathway, a high-throughput cell-based screening model based on dual luciferase reporters system was established, a number of genes that can activate NF-κB signal pathway were obtained by screening of 439 novel function genes. Among them, TMEM9B can obviously activate NF-κB signaling pathway. Further experiments showed that TMEM9B activated NF-κB signaling pathway in a dose-dependent pattern. Western blotting and EMSA experiments confirmed that TMEM9B can promote the degradation of IκBα (a cytoplasm inhibitor of NF-κB), and cause NF-κB shift from the cytoplasm to nucleus. At the same time, flow cytometry result demonstrated TMEM9B can induce apoptosis in HEK293T and HeLa cells. In short, a stable and effective screening system for NF-κB has been established, through which TMEM9B was identified to be able to significantly activate NF-κB signal transduction pathway and thus cause cells apoptosis.

Construction and identification of plasmid with luciferase reporter gene for detection of T-bet expression activity / 中华微生物学和免疫学杂志

Objective To construct a T-bet response reporter gene, for the detection of T-bet tran-scriptional activity and application in high-throughput screening for the functional genomies. Methods The cis-acting DNA element, ThRE, based on CNS-22 T box site of IFN-γ gene, was recombined into a reporter vector pLUC-MCS. The reporter gene was transfected into HEK 293T cells to detect its response to T-bet. And the binding of T-bot to TbRE was identified with electrophoretic mobility shift assay(EMSA). Results ThBE was successfully cloned into pLUC-MCS, named as TbRE-LUC. Using a luciferase assay, expression of the reporter gene is found to be induced by T-bet in a dose dependent manner and correlate with T-bet ex-pression positively with activation up to 20 folds. Moreover, the binding specificity of T-bet to TbRE is vali-dated by EMSA. Conclusion We successfully constructed a T-bet response reporter gene, ThRE-LUC, which responds to T-bet keenly and specifically. TbRE-LUC will be a useful tool in high-throughput screen-ing for human genes associated with transcription activity of T-bet.

Experimental Study on the Anti-inflammation Effect of 3,4-oxo-isopropylidene-shikimic Acid / 中国中医药信息杂志

Objective To study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on transcription factor STAT1, STAT3 and NF-?B in human cervix cancer Hela cell. Methods The cell proliferation was assessed by MTT assay. The luciferase activity of transcription factor STAT1, STAT3 and NF-?B was determined by the Dual-Luciferase Reporter Assay System. Results ISA can not inhibited the growth of Hela cell. The luciferase activity of transcription factor NF-?B in Hela cells treated with ISA was inhibited, while the luciferase activities of transcription factor STAT1 and STAT3 were not inhibited. Conclusion ISA can inhibit inflammation, which may be related with suppression of NF-?B transcriptional activity.