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. 2015 Aug 13;5:12983. doi: 10.1038/srep12983

Figure 8. Bidirectional regulation of Nrf1 by proteasome- and/or calpain inhibitors to control target gene expression.

Figure 8

(a) After 6-h transfection with Nrf1, GSTA2-6 × ARE-Luc and β-gal constructs, COS-1 cells were allowed to recover overnight in 5.5 mM-glucose medium before being treated for 18 h with MG132, CI/ALLN), CII, or CP at the indicated doses (μmol/l for MG132; μg/ml for the other three chemicals) in 5.5 or 25 mM-glucose medium. Thereafter, Nrf1-mediated activity (upper) was measured and is shown as a fold change against the vehicle value (designated 1.0). By comparison with the activity obtained from cells grown in medium containing 25 mM-glucose, significant increases ($p < 0.05; $$p < 0.001, n = 9) or significant decreases (*p < 0.05 and **p < 0.001, n = 9) in reporter activity are indicated. The abundance of Nrf1 isoforms was examined by immunoblotting, and the results are shown from cells cultured in 25 mM-glucose (middle) or 5.5 mM-glucose media (Fig. S6B). (b) Bidirectional regulation of Nrf1 by proteasome and calpain inhibitors as assessed by measuring PMSA4/ARE-driven reporter activity. COS-1 cells expressing Nrf1, 3 × PMSA4-ARE-Luc (3 × PMSA4-mutARE-Luc as a negative control)3,30 and β-gal, were treated with the indicated doses of chemicals, followed by statistical analysis of luciferase activity, as described above. (c) Dual regulation of a CAT reporter gene by CI/ALLN and MG132. COS-1 cells expressing Nrf1, the reporter pCAT1500 (which contains 1500 bp of the promoter of PMSB6 ligated to the CAT gene2; the ARE-Mut17 served as a negative control) and β-gal, were treated with the indicated doses of chemicals as described above. CAT activity was measured and compared against that for β-gal activity. (d) The cytotoxic effect of chemicals on COS-1 cells was assayed as described in the Materials and Methods section. (e) The activity of Nrf1 in response to glucose starvation was determined using the GSTA2-6 × ARE-Luc reporter assay, after an 8-h recovery of the transfected cells in 5.5 mM glucose-containing medium, and an additional 18-h during which time they were grown in medium containing the indicated glucose concentrations. Ectopic Nrf1-forced reporter gene activity was calculated as a ratio of its value against the background activity mediated by endogenous Nrf1 and/or Nrf2 (i.e. pcDNA3).