Biotinylated-psoralen (bPsoralen) incorporation was employed to evaluate (−) DNA supercoiling, following the methodology outlined in 43 (link). Briefly, cells were cultured on polylysine pre-treated glass slides and exposed to 75 μM bPsoralen (EZ-Link Psoralen-PEG3-Biotin, ThermoFisher) along with 0.01% digitonin (Sigma, D141) for 20 minutes at room temperature in dark conditions. After 10 minutes of UV crosslinking at 360 nm, slides were fixed in 4% PFA-PBS for 15 minutes at room temperature. Then, slides were blocked in 5% BSA-PBS before incubating with primary antibodies (1:2500 α-ERα mouse antibody and 1:1000 α-biotin) for 30 minutes. Coverslips were then washed three times in 0.1% Tween20-PBS and incubated with secondary antibodies for 30 minutes. DAPI counterstaining and ProLong mounting (ThermoFisher) were performed for image acquisition using a LEICA confocal microscope SP5. Quantification of bPsoralen was restricted to nuclei (DAPI) or intranuclear regions exhibiting a high density of ERα signal (HDR). As negative controls, cells were pretreated with triptolide (10 μM, 5 h).