Fig. 4

SUMOylation dictates the chromatin binding of AR. a PLA shows predominant nuclear localization of SUMOylated AR when a hyperSUMO environment is introduced. MCF-7 cells were co-transfected with AR + EV or SUMO-3. The DuoLink kit was used according to manufacturer’s instructions and total number of AR/SUMO complexes (red puncta) per nuclei were quantified using Image J. Represented images were derived from original images by deconvolution; white-dashed lines highlight the nuclei. b Graph represents quantification of nuclear red puncta as mean ± SEM. Student’s t-test was used to compare groups and ***p < 0.001 was considered statistically significant. c SUMO-3 enhances SUMOylation and recruitment of AR to chromatin even when cells were unstimulated with synthetic androgens (R1881). ET-R-like conditions were introduced in hormone-deprived MCF-7 cells by overexpressing AR with and without SUMO-3. After 24 h, cells were unstimulated or stimulated with 10 nM R1881. Chromatin-bound AR were immunoprecipitated, resolved by SDS-PAGE and immunoblotted with specific antibodies. SUMO-2/3 conjugates of AR are identified with red arrows