Figure 2. CLE is necessary to induce transcriptional response to MG132.

(A) U-251 MG cells were transfected with 5′-deleted fragment of the clusterin promoter, and treated for 16 h with MG132 (5 μM). Luciferase activities were measured and normalized to the β-galactosidase (b-gal) activities. The fold of MG132-induced transcription is indicated for each construct. a.u., arbitrary units. (B) U-251 MG cells were transfected with the pClu-218bp clusterin promoter or with the same DNA fragment containing a mutated CLE sequence (pClu-218bp mut.CLE). As control, constructs containing the αB-crystallin promoter were used, either with a wild-type (pαBcryst) or with a mutated HSE (pαBcryst mut.HSE). Heterologous promoters containing the wild-type (CLE) or the mutated (mut.CLE) CLE in front of the control promoter TK were also assayed. One, two or four repeats of CLE were inserted (CLE_x1-TK, CLE_x2-TK, mut.CLE_x2-TK and CLE_x4-TK respectively). As a positive control, we used a tandem repeat of a consensus HSE in front of a luciferase reporter gene (HSE_x2-TATA). After transfection, cells were treated with MG132 or untreated. Results are shown as the fold induction and represent the means±S.E.M. for four to ten independent experiments.