Figure 2
From: Ets-1 mediates upregulation of Mcl-1 downstream of XBP-1 in human melanoma cells upon ER stress
An Ets binding region between −205 and −175 of the Mcl-1 promoter is transcriptionally responsive to ER stress. (a) A schematic illustration of a potential c-Rel and Ets-1 binding motif located within the −250/−175 region of the Mcl-1 promoter. The Ets consensus binding motif is boxed in highlighted in gray. Experimental mutagenesis of the region is also depicted. (b) Mel-CV and MM200 cells were transiently transfected with the pGL3-basic-based reporter constructs, pGL3-vector, pGL3-Mcl-1−205/+10 (pGL3-Mcl-1), and a reporter with the c-Rel binding region mutated as shown in (a) (pGL3-Mcl-1-mut), respectively. After 24 h, cells were treated with TM (3 μM) for a further 16 h followed by measurement of the luciferase activity. (c) Formaldehyde-cross-linked chromatin of Mel-CV and MM200 cells with or without treatment with TM (3 μM) for 16 h were subjected to immunoprecipitation with antibodies against Ets-1, Ets-2 and c-Rel, respectively. The precipitates were subjected to PCR amplification using primers for the −205/+10 region of the Mcl-1 promoter. (d) Nuclear extracts of MM200 cells with treatment with TM (3 μM) for 16 h were subjected to super shift assay. Comp: competitor; NE, nuclear extracts; SC, specific competitor. E, the complex formed in the presence of nuclear extracts with labeled oligonucleaotides. Bars, s.e. (n=3).
