Fig. 3. The effect of inhibition of intracellular androgen receptor on testosterone-induced Ca²⁺ oscillations. (A) Fluorescence images of iAR localization were superimposed on bright-field images of single cells. (Upper panel) In control cells, iAR was distributed throughout the cell, but there was a higher relative abundance in the nucleus. Note also the clusters of iAR in the neurites. (Lower panel) Upon testosterone (100 nM) stimulation for 1 hour, the cytoplasmic iAR translocated to the nucleus. (B,C) Cells were transiently transfected with AR-siRNA and the expression of the protein was reduced 80% with respect to control conditions, ^*P<0.05 versus basal (n=4). (D,E) Neither iAR-siRNA nor cyproterone modify the ability of testosterone to induce Ca²⁺ oscillations. (F) To show that internalization of testosterone is not required, the plasma membrane-impermeable testosterone bound to albumin (T-BSA) was tested and it mimicked the effects of the free hormone.