Bay11 7082 significantly attenuated the increased transcriptional activ ity of NF ��B driven sellekchem luciferase reporter in these two cell lines, thus confirmed the efficiency of Bay11 7082 as an NF ��B inhibitor. Notably, the increased tran scriptional activity of the Mcl 1 promoter observed in Eca109 cells remained unchanged by the above three strategies. Taken together, these results pro vide consistent evidence that the involvement of NF ��B pathway in the Mcl 1 promoter transcriptional activity in various human ESCC cells. NF ��B signaling pathway contributes to Mcl 1 expression in various human esophageal squamous cell carcinoma Inhibitors,Modulators,Libraries cell lines We further confirm whether NF ��B is involved in Mcl Inhibitors,Modulators,Libraries 1 expression in human ESCC cells. Bay11 7082 was firstly used to investigate the effect of NF ��B activation on Mcl 1 induction.
Treatment of TE 1 cells with the in hibitor resulted in a dose dependent attenuation of Mcl 1 induction. Similar results were obtained Inhibitors,Modulators,Libraries from KYSE150 cells treated with various concentrations Mcl 1 ��Bwt generated higher luciferase activity than that of the pGL2 Basic construct, indicated that high transcrip tional activity of human Mcl 1 promoter in three Mcl 1 expressing ESCC cell lines tested. However, with a pro moter construct mutated at the ��B site, the loss of Mcl 1 promoter activity was observed in TE 1 and KYSE150 cells. Dominant negative mutants of I��B, a truncant mutant with a deletion of 71 amino acids at the N terminus of I��B, can competitively inhibit the activation of NF ��B was used to block NF ��B activation Inhibitors,Modulators,Libraries as described previously.
Expression of DNMI��B significantly inhibited the Mcl 1 promoter ac tivity in TE 1 and KYSE150 cells. Further more, compared with their respective DMSO control, treatment with 20 uM Bay11 7082, a specific NF ��B in hibitor, resulted in the Mcl 1 promoter activity drastically curtailed in both TE 1 and KYSE150 cells. The activity of the Inhibitors,Modulators,Libraries Mcl 1 promoter with mutated NF ��B site was essen tially unaffected by inhibitor treatment. NF ��B transcriptional activities in both TE 1 and KYSE150 cell lines have also been estimated by using an NF ��B of Bay11 7082. DNMI��B was further used to test the role of NF ��B pathway in regulating Mcl 1 expression. As verified by Western blotting analysis, ex pression of DNMI��B in TE 1 or KYSE150 cells led to a significant decrease of Mcl 1 induction compared with the vector control.
The results suggested GW572016 that NF ��B pathway is involved in Mcl 1 ex pression in TE 1 and KYSE150 cells. Binding of transcription factor NF ��B family members to human Mcl 1 promoter To ascertain whether NF ��B transcription factor can bind the NF ��B site in human Mcl 1 promoter, EMSA was performed with an oligonucleotide probe containing the putative NF ��B binding sequence derived from hu man Mcl 1 promoter.