Cell culture

Human MCF7 breast cancer cells (a kind gift from Prof. Dr. Pernette Verschure, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands), Human T47D and T47DS breast cancer cells (a kind gift from Stieneke van den Brink, Hubrecht institute, Utrecht, The Netherlands), and HEK293TN cells (System Biosciences, #LV900A-1) were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing GlutaMAX (Gibco, #11584516), supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, #11573397). Cells were split 1:5–1:10 twice a week and routinely tested for mycoplasma. MCF7 cells transfected with a PR expression construct are referred to as MCF7 (+ PR). MCF7 cells stably expressing a lentiviral PR construct are referred to as MCF7/PR.

Immunofluorescence staining and fluorescence microscopy

One day prior to imaging, 75,000 MCF7 or 100,000 T47D cells were seeded on an 8-well chamber slide with glass bottom (Ibidi, #80827–90). The next day, cells were treated with 20 nM R5020 (Promegestone, Perkin Elmer, #NLP004005MG), or 20 nM R5020 and 100 nM RU486 (Mifestrone, Sigma Aldrich, #475838) dissolved in ethanol, which was also taken along as a negative control. Two hours after treatment, the cells were fixed in 4% paraformaldehyde (Alfa Aesar, #43368) in PBS for 15 min at room temperature (RT) and washed with HBSS (Hanks' Balanced Salt Solution, (Thermo Fisher Scientific #11550456) three times. Next, the cells were permeabilized with 0.1% Triton in PBS for 15 min at RT. After three washes with HBSS, the samples were blocked for 2 h in HBSS with 4% BSA (Tocris BioScience, #5217). Incubation with primary antibody rabbit anti-progesterone receptor (1:500 in 4% BSA, Cell Signaling #8757/D8Q2J, recognizing both PR-A and PR-B) was performed overnight (O/N) at 4 ̇C. Following three washes with HBSS, the cells were incubated for 2 h with secondary antibody AlexaFluor 488 Goat Anti-Rabbit IgG (1:1000 in 4% BSA, Invitrogen #A11008) at RT in the dark. The samples were stained with DAPI (1:1000 in HBSS, Invitrogen, #D1306) for 10 min at RT, washed three times with HBSS and imaged on an SP8 confocal microscope (Leica Microsystems). Imaging was performed using a 63 × oil objective with 405 (5% laser power) and 488 (8% laser power for MCF7 and 2% laser power for T47D) lasers, using a PMT1 detector (gain 700) for fluorescent signal with a 413–469 bandpass for DAPI and a HyD detector (gain 100) for fluorescent signal with a 496–547 bandpass for GFP. Images for the individual channels were extracted using Fiji [52]. Care was taken to image each sample with the same settings. For image analysis, Cell Profiler [53] was used to segment the nucleus based on the DAPI signal, excluding border nuclei. The nuclear mean intensity of the PR staining was then measured for each individual cell, normalized to the average intensity in the non-treated condition and plotted in GraphPad Prism (Version 10.0.0).

DNA cloning

The 2xPRE-luciferase reporter (2X PRE TK luc) construct contains 2 consensus PRE sites upstream of a minimal thymidine kinase (TK) promoter (Table 2, #1). The pcDNA3-PRB plasmid contains the full-length PR sequence (Table 2, #2). To generate PRE-luc constructs containing concatemerized, wildtype or mutated PRE sites, primers were designed using Snapgene (Table 1). The PRE-luc constructs were generated using restriction cloning of the annealed oligonucleotides into the 2xPRE-luc vector with BamHI (Thermo Fisher Scientific, #FD0054). For the vectors with multiple PRE sites, restriction enzyme digestion and ligation were repeated until constructs with 4, 6, 8, 10 and 12 × PRE sites were obtained.

For generation of lentiviral PR expression vectors, full-length PR was cloned into the multisite gateway compatible pMuLE-ENTR-MCS-R4-R3 (Table 2, #3) by restriction cloning from pcDNA3-PRB (Table 2, #2). For generation of entry clones containing 2x, 4x, or 6xPRE sites for multisite gateway reactions, annealed oligonucleotides coding for the 2YBTATA minimal promotor [54] (Table 1) were ligated into the pMuLE ENTR MCS L1-L4 vector (Table 2, #4), followed by ligation of annealed 2xPRE oligonucleotides (Table 1).

Lentiviral 2/4/6xPRE-GFP and CMV-PR plasmids were generated using multisite LR gateway reactions. Gateway vectors were diluted to 10 fmol/ul of each entry plasmid and 20 fmol/ul of the destination plasmid with TE buffer (pH 8.0). Lenti-CMV-PR constructs were created by mixing 10 fmol 5’ entry CMV promoter (Table 2, #5), 10 fmol pMuLE (R4-R3) PRB, 10 fmol 3’ entry polyA (Table 2, #6), and 20 fmol destination vector Lenti-Blast (Table 2, #7). For Lenti-2/4/6xPRE-GFP reporters: 10 fmol 5’ entry plasmids containing 2/4/6xPRE and 2YBTATA, 10 fmol middle entry eGFP (Table 2, #8), 10 fmol 3’ entry polyA (Table 2, #6), and 20 fmol destination vector Lenti-DEST (Table 2, #9) were combined. LR Clonase II Plus enzyme (Thermo Fisher Scientific, #12538120) was added to catalyze the gateway reactions according to the manufacturer’s instructions.

Dual luciferase assays

MCF7, MCF7/PR, T47D or T47DS cells were plated in 24-well plates at a density of 100,000 (MCF7/MCF7/PR) or 150,000 (T47D/T47DS) cells per well. After 24 h, the medium was replaced with DMEM supplemented with 5% charcoal stripped FBS (Thermo Fisher Scientific, #A3382101), or one of the following four media for optimization of medium conditions: 1) DMEM supplemented with 10% FBS (identical to the standard propagation media), 2) DMEM supplemented 5% charcoal stripped FBS (stripped FBS – the final media used for all experiments after Fig. 2c), 3) phenol red free (PRF) DMEM (Thermo Fisher Scientific, #11594416) supplemented with 10% FBS or 4) phenol red free DMEM supplemented with 5% charcoal stripped FBS. Cells were transfected using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich, #06366546001), according to the manufacturer’s instructions. For MCF7, transfections were performed in duplicate, using a total amount of 500 ng plasmid DNA per well, consisting of 200 ng Luciferase construct and 200 ng PR expression vector (Table 2, #2) and 100 ng Renilla construct. For MCF7/PR, T47D and T47DS 400 ng Luciferase construct and 100 ng Renilla construct was used. On the next day, cells were treated with either ethanol (EtOH, control), 20 nM R5020 (Perkin Elmer, #NLP004005MG), or 20 nM R5020 and 100 nM RU486 (Sigma Aldrich, #475838) in fresh medium. When comparing different R5020 concentrations, the cells were treated with the indicated range of R5020 (1 pM – 100 nM) or EtOH as the solvent control. To determine reporter specificity, the cells were treated with 10 nM of the following compounds: R5020, β-estradiol (Sigma, #E8875-1G), aldosterone (Sigma-Aldrich, #A9477-5MG) dexamethasone, (Sigma-Aldrich, #D4902), dihydroxytestosterone (Merck, #D-073) or EtOH as solvent control. Cells were lysed after exactly 24 h of stimulation in 1X Passive Lysis Buffer (100 µl per well (Promega, #E1941)). For the dual luciferase measurements, non-commercial firefly, and Renilla Luciferase Reagents (LAR) were used [60]. Firefly and Renilla luciferase activity was measured in a GloMax Navigator (Promega, #GM2000). Firefly luciferase values were normalized to Renilla luciferase values. The data is presented as fold-change in firefly luciferase activity, normalized over the non-treated control, unless stated otherwise. Plots are generated using GraphPad Prism. (Version 10.0.0).

qRT-PCR

MCF7, MCF7/PR, T47D and T47DS cells were plated in 6-well plates at a density of 300,000 (MCF7/ MCF7/PR) or 400,000 (T47D/T47DS) cells per well. The following day, for medium was refreshed for all cells and MCF7 cells were transfected with 2 µg pcDNA3-PRB per well using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich, #06366546001) according to the manufacturer’s instructions. 48 h after plating, cells were treated with EtOH, 20 nM R5020 (Perkin Elmer, #NLP004005MG), or 20 nM R5020 and 100 nM RU486 (Sigma Aldrich, #475838). When comparing different R5020 concentrations, the cells were treated with 1 pM – 100 nM R5020 or EtOH. After 24 h treatment, RNA was isolated using Trizol according to the manufacturer’s instructions. Total RNA was DNAse treated with RQ1 DNAse (Promega, #M6101). cDNA synthesis was performed using 4000 ng RNA using SuperScript IV Reverse Transcriptase (Invitrogen, #18090200) and Random Hexamers (Invitrogen, #N8080127) according to manufacturer’s guidelines with the addition of RiboLock RNase Inhibitor (Thermo Fisher Scientific, #EO0328). cDNA was diluted tenfold and qRT-PCR reactions were performed using 5 × HOT FIREpol EvaGreen qPCR mix plus (ROX) (Bioconnect, #08–24-00020) and a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). qRT-PCR primers were thoroughly checked to have a single melt curve. Primer sequences are listed in Table 3 and 4 Calculations were performed using the ddCt method and presented as relative values normalized over YWHAS and EtOH treated conditions. Plots are generated using GraphPad Prism (10.0.0).

Table 3 Generated plasmids (deposited with Addgene)Western Blot

For Western Blot analysis, the cells were plated and treated in 6-well plates and lysed using 100 µl lysis buffer (20 µM Tris pH 8.0, 2 µM EDTA pH 8.0, 0.5% NP40, 25 µM sodium B-glycerophosphate, 100 µM Sodium fluoride, 10 mM sodium pyrophosphate). Protein concentrations were measured using the Pierce BCA protein Assay kit (Thermo Fisher Scientific #23225) and a total of 30 µg of protein for each sample was loaded on a 10% SDS-PAGE gel. Proteins were transferred on a 0.2 µm nitrocellulose membrane using the trans-blot turbo transfer system (Bio-Rad) and blocked with 1:1 diluted TBS Odyssey Blocking buffer (LI-COR Biosciences, #927–50100). Primary antibody directed against PR (1:1000, Thermo Fisher Scientific #MA5-16393, recognizing both PR-A and PR-B) and Actin (1:1000, MP biomedicals #08691001) were diluted in blocking buffer supplemented with 0.1% Tween-20. Primary antibody staining was performed O/N at 4 °C followed by incubation with secondary antibodies (1:20,000 IRDye 680L, #926–6802 or 1:20,000 IRDye 800CW LI-COR, #926–32211), in TBS supplemented with 0.1% Tween-20 for one hour at RT and detection was performed at 700 nm and 800 nm using an Odyssey Fc (LI-COR Biosciences).

Generation of MCF7/PR and MCF7/PR-2/4/6xPRE-GFP lines

For lentivirus production, 5 × 106 HEK293TN cells were plated in 10 cm plates. The next day, the cells were transfected with 3 µg packaging vector pCMVDR8.2 (Table 2, #10), 3 µg RSV-rev (Table 2, #11), 3 µg VSVg (Table 2, #12), and 8 µg custom generated lentiviral CMV-PR or 2x/4x/6xPRE-GFP plasmids using PEI (Polyethylenimine, Polysciences, #23966). Medium was refreshed after 24 h, virus was collected after 48 h, filtered through a 45 µm filter, and diluted 1:4. MCF7 cells were infected in the presence of polybrene (1:2000, Merck Millipore #TR-1003-G). After 24 h incubation, cells were, split, and selected for up to two weeks with blasticidin (10 µg/ml Thermo Fisher Scientific, #11583677) or G418 (700 µg/ml Gibco, #11811–031).

Fluorescence-activated cell sorting (FACS)

For FACS experiments, MCF7/PR-2/4/6xPRE-GFP cells were plated in 6-well plates for analysis, or 10 cm plates for sorting, in fresh DMEM supplemented with 5% stripped FBS, 48 h prior to analysis or sorting. At 24 h, the cells were treated with EtOH, 20 nM R5020 (sorting) or a R5020 concentration range from 10 pM to 100 nM (analysis) in fresh DMEM medium supplemented with 5% stripped FBS. 24 h after treatment, the cells were trypsinized, and pelleted. The cells were stained with 1 µg/ml DAPI (Invitrogen, #D1306) in HF (2% stripped FBS in HBSS), washed and again resuspended in HF and then filtered through a 70 µm filter. Sorting and analysis were performed on a FACSAria™ III (BD, Franklin Lakes, NJ). For FACS sorting, the cells with intermediate GFP expression levels were sorted in 24-wells plates containing full medium + 1% penicillin/streptomycin and 0.025 M HEPES. Analysis of FACS results was performed using Flowjo (10.8.2). Gates for GFP positive cells were set using MCF7 wildtype and EtOH treated samples of 2xPRE-GFP. The gating strategy was expanded to all other samples.

Imaging of PRE-GFP lines

For imaging, MCF7/PR-2/4/6xPRE-GFP cells were seeded on an 8-well chamber slide with glass bottom (Ibidi, #80827–90) containing DMEM medium supplemented with 5% stripped FBS. After 6 h, medium was replaced with phenol red free DMEM supplemented with 5% stripped FBS, containing either EtOH or 20 nM R5020. The next morning, phenol red free DMEM supplemented with 5% stripped FBS, 0.025 M HEPES (Thermo Fisher Scientific, #11560496), and 500 nM SiR-DNA was added and 4 h later, cells were imaged at 37 °C on an SP8 confocal microscope (Leica Microsystems) using a 25 × water objective, 488 (8% laser power) and 633 (8% laser power) lasers, using a HyD detector (100 gain) for fluorescent signal with a 496–559 nm bandpass for GFP and a PMT3 detector (700 gain) for fluorescent signal with a 642–693 nm bandpass for SiR-DNA. Images were processed using Fiji [52].

Statistical analysis

For quantification of PR nuclear abundance (Fig. 1), p-values were calculated using a one-way ANOVA followed by a Tukey’s, Šídák's or Dunnett’s multiple comparison test. For luciferase assays and qRT-PCR analyses, p-values were calculated using a 2-way ANOVA followed by a Tukey’s multiple comparison test, except for the analysis of PGR expression (SupFig. 1) where a one-way ANOVA followed by a Tukey’s multiple comparison test was used. All statistical analyses were performed and plotted using GraphPad Prism (10.0.0).