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. 2020 Apr 25;12(5):1065. doi: 10.3390/cancers12051065

Figure 2.

Figure 2

Nelfinavir inhibits Nrf1 transcriptional activity by a dual mode of action. (A) Left: HEK293 cells stably expressing 3×PSMA4-ARE-Luc reporter were transfected with renilla luciferase gene for normalization and co-treated with 1 μM MG132 and nelfinavir concentrations spanning 1 μM to 20 μM. At 16 h post-transfection, dual luciferase assays were used to measure firefly and renilla luciferase activity. Normalized luciferase activity is shown. The data were fitted by sigmoidal and bi-sigmoidal functions, and IC50 values were calculated. Right: U2OS cells stably expressing UbG76V-GFP reporter were treated with nelfinavir at concentrations spanning 1 μM to 20 μM. The GFP fluorescence (dependent on proteasome activity) was measured at different time points (0–24 h), and maxima were measured. The data were fitted by sigmoidal and bi-sigmoidal functions, and EC50 values were calculated for the bi-sigmoidal fit. (B) HEK293 cells were co-treated with 200 nM BTZ and 10 μM or 20 μM NFV or DMSO for 16 h. TCF11/Nrf1 was detected using monoclonal antibody D5B10 (Cell Signaling, #8052; 1:50). The amount of nuclear TCF11/Nrf1 was estimated as integrated fluorescent nuclear intensity obtained by CellProfiler processing [29].