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. 2017 Jul 6;7:4835. doi: 10.1038/s41598-017-04838-8

Figure 2.

Figure 2

Δ6 MR is a ligand-independent transactivator and exerts dominant-negatif effects on FL MR. (a) Analysis of subcellular trafficking of the Δ6 MR splice variant. For immunocytochemistry, COS7 cells were cultivated in 4-well chamber slides (Sarstedt). Cells were transfected with 500 ng of FL MR- or Δ6 MR-encoding vector in the presence of Lipofectamine 2000 and incubated for 1 hour with ethanol (vehicle) or 10 nM aldosterone (Aldo). Cells were fixed and processed for MR immunodetection with the 39 N anti-MR antibody. Representative images of the subcellular localization of MR FL and MR Δ6 before and after hormone treatment. After aldosterone stimulation, FL MR translocated to the nucleus, whereas Δ6 MR was already present in the nucleus, even in the absence of the ligand. The nuclei were stained with DAPI (blue). (b) HuR immunodetection was coupled with an automated high-throughput microscopy (HTM) analysis, to quantify the subcellular trafficking of this protein. The results are expressed as the mean ratio of nuclear to cytoplasmic fluorescence ± SEM (n > 800 cells). Bar, 25 µm. ***P < 0.001; NS = not significant (Mann-Whitney U-tests). (c,d,e) HEK 293 T cells were transfected as described in the Materials and Methods. (c) HEK 293 T cells were transfected with various amounts of the plasmid encoding Δ6 MR (0 to 80 ng). The following day, firefly-luciferase activities were measured and normalized relative to β-galactosidase activities. The data shown are means ± SEM of two independent experiments (n = 24). ***P < 0.001 (Mann-Whitney U-tests). (d) After transfection, HEK 293 T cells were incubated for 24 hours with ethanol (vehicle) or with 1 nM aldosterone. Firefly-luciferase activities, normalized to β-galactosidase activities, are means ± SEM (n = 8). ***P < 0.001; ### P < 0.001 (Mann-Whitney U-tests). (e) Δ6 MR acts as a dominant-negative transactivator of FL MR. HEK 293 T cells were transfected with FL MR alone or with a one-fold (20 ng) or two-fold excess (40 ng) of Δ6 MR. Cells were incubated for 24 hours with ethanol (vehicle) or with 0.1 or 1 nM aldosterone. Luciferase activities were measured as described above. Data are means ± SEM of three independent experiments (n = 24). ***P < 0.001; ### P < 0.001 (Mann-Whitney U-tests).