Figure 7: ERα nuclear translocation and colocalization with Icaritin.

Icaritin uptake by mESCs at the indicated time points was detected by confocal microscopy using excitation wavelength 350 nm (a) and flow cytometric analysis (b) respectively. (c) Translocation of ERα protein into the nucleus was visualized by immunostaining with ERα antibody and nuclear counterstain by PI. (d) Time-dependent ERα protein nuclear translocation was detected by Western blot analysis of protein extracted from cytosolic and nuclear fractions. Shown are cropped from the original blots. (e) Representative immunofluorescent images showing colocalization of Icaritin and ERα protein in mESCs upon 24 h of drug exposure. (f) Representative Icaritin/ERα colocalization (line a and b) in mESCs shown in (e). Line profiles indicated the extent of colocalization over distance. Average Pearson’s Correlation analyses indicated high levels of colocalization of ERα and Icaritin. (g) A direct interaction of Icaritin with ERα was validated by protein thermal shift assay. Experiment was performed in the absence or presence of ERα recombinant protein with an indicated dose of Icaritin. The resulting ΔTm values were analyzed by thermal protein shift software. Values were the mean ± SD (n = 4). **P < 0.01.