Figure 4.

DXM-induced nuclear translocation of the WT and the six GR variants. COS-7 cells (grown in F6 or 100-mm petri dish flasks) were transiently transfected with 400 ng of plasmid encoding the WT or the six GR variants (F65V, M86V, A229T, A304E, N374S, and R386Q). After overnight recovery, cells were stimulated by ethanol (−) or 100 nM DXM (+) for 1 h and fixed and processed for immunofluorescence studies, followed by quantification analysis with high-throughput microscopy (see Methods section). (A) Representative images of cyto-nuclear translocation of the WT and F65V GR variant with ethanol (control) or with DXM for 1 h. Images obtained by deconvolution microscopy at 60× magnification and nuclear staining by 4′,6-diamidino-2-phenylindole (blue) and GR (red) using antibody [29] from Santa Cruz Biotechnology. White bar, 20 µm. (B) Comparison of the cyto-nuclear shuttling of the six variants compared with that of the WT after quantification by high-throughput microscopy on >1500 transfected cells per condition. Results are expressed as mean ± SEM. Nuclear-cytoplasmic ratios for each construction were normalized to the mean value arbitrarily set at 1 under basal conditions (i.e., in the absence of DXM stimulation). Statistical analysis was performed by an ANOVA test followed by Bonferroni posttests. ***P < 0.001. ns, not significant.