FIG. 2.
Cytoplasm-nucleus translocational oscillations of Nrf2 in human vascular endothelial HMEC-1 cells in vitro and response to functional stimulation. (A) Fluorescence microscopy image frames of HMEC-1 cells expression GFP-Nrf2 showing oscillation of cytoplasm-nucleus translocation. Images frames taken at time 46, 168, 218, 280 and 316 min poststimulation of HMEC-1 cells with 2 μM SFN. (B, C) Typical time course changes in GFP-Nrf2 nuclear/cellular localization ratio for control cells and cells stimulated with 2 μM SFN, respectively. The ratio plotted is ĪNucleus/ĪCell where ĪNucleus and ĪCell are average pixel intensities of GFP-Nrf2 fluorescence of the nucleus and whole cell in the field of view, respectively. NB ĪNucleus/ĪCell relates to the ratio of total nucleus/cell GFP-Nrf2, r, by the equation: r=(ĪNucleus×ANucleus)/(ĪCell×ACell), where ANucleus and ACell are areas of the nucleus and whole cell, respectively. Ratio r varies from 0 to 1 and ĪNucleus/ĪCell from 0 to >1. (D) Cumulative frequency distribution of GFP-Nrf2 cytoplasm-nuclear translocation oscillation period (histogram distribution shown inset). Median (lower–upper quartile): control - - -, 129 (81–175) min (n=44); +2 μM SFN —, 80 (64–128) min (n=53), p<0.001; Mann–Whitney U test. (E) Cumulative frequency distribution of GFP-Nrf2 cytoplasm-nuclear translocation amplitude (histogram distribution shown inset). Median (lower–upper quartile): control ---, 0.65 (0.35–0.87) arbitrary units (n=44); +2 μM SFN —, 0.40 (0.34–0.59) arbitrary units (n=53), p<0.05; Mann–Whitney U test. (F) Time course of NQO1-ARE transcriptional activity in control (□–□) and SFN stimulated (■—■) HMEC-1 cells in vitro. Data are mean±SEM, n=11; significance—***p<0.001; t-test. Dose response of NQO1-ARE transcriptional response on activator concentration in HMEC-1 cells studied by luciferase reporter assay. Luminescence output is given in RLU. (G, H) Six-hour-treatment-response data were fitted to a logistic regression equation, NQO1-ARE transcriptional response=[Activator]n/([Activator]n+EC50n)+c, where EC50 is the median effective concentration, n the logistic regression coefficient or Hill coefficient, and c the constitutive or background response. (G) SFN, EC50=1.3±0.3 μM, n=1.64±0.58 and c=29.3±3.3 (n=13); and (H) QTN, EC50=8.3±0.5 μM, n=3.81±0.79, c=32.3±3.3 (n=11). Correlation of oscillation amplitude on period. (I) Control cells—correlation coefficient r=0.63 with regression equation: amplitude=0.0028×Period (min)+0.27; p<0.001. (J) Cells activated with 2 μM SFN−correlation coefficient r=0.62 with regression equation: amplitude=0.0035×Period (min)+0.17; p<0.001. GFP, green fluorescent protein; HMEC-1, human microvascular endothelial cell line-1; RLU, relative light unit. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars