Data sources

We collected the associated gene expression profiles in publicly available Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Samples from different menstruation phases (proliferative/PE, early secretory/ESE, mid-secretory/MSE, late secretory/LES) were chosen from GSE4888 and GSE56364 to detect expression of PGRMC1 [21, 22]. All raw data were background-subtracted and normalized.

Cell culture

The hTERT-immortalized human endometrial stromal cells (T-HESCs) were purchased from abm (T0533). Both the cell lines T-HESCs and St-T1 were maintained in phenol-red free Dulbecco’s Modified Eagle Medium//Ham’s F12 (DMEM/F12; Gibco, Thermo Fisher Scientific, 11039021) medium supplemented with 10% (v/v) charcoal-stripped fetal bovine serum (Thermo Fisher Scientific, 12676029), 100 units/mL penicillin–streptomycin (Thermo Fisher Scientific, 2321118), 50 µg/ml gentamycin sulfate (Biowest, L0012), 200 µM sodium pyruvate (Biowest, L0624) and 1.5 g/L sodium bicarbonate (Biowest, L0680) (hereafter referred to as complete medium) in a humidified incubator at 37 °C in the presence of 5% CO2. Cells (passage number < 10) were regularly tested negative for mycoplasma.

Chemical compounds

AG205 (Sigma-Aldrich) was diluted in 2% charcoal-stripped FBS complete medium to 15 mM. Medroxyprogesterone acetate (MPA) and 8-Br-cAMP MPA (cAMP) were prepared from a 10 mM and 5 mM stock solution, respectively.

MTT Assay

We measured activated cellular metabolism as a surrogate for proliferation by performing the MTT assay. Briefly, T-HESCs cells (5 X 103 cells per well) were seeded in triplicates in 96-well plate in complete medium and grown for 24 h. After the attachment, cells were either treated with or without induction cocktail in decidualization medium. On the day of assay, cells were incubated with 0.25 mg/ml MTT (Sigma-Aldrich) in decidualization medium for 3 h at 37 oC. Following 1 h of incubation with DMSO at 37 oC and 300 rpm in a microplate shaker, absorption was measured at 540 nm using TECAN Spark® spectrophotometer.

Immunofluorescence staining

Cells were seeded and cultured in chamber slides (Nunc Lab-Tek, Thermo Fisher Scientific C7182-1PAK) fixed with 4% formaldehyde (Sigma-Aldrich, 20649296018) for 10 min at room temperature (RT), washed with washing buffer (Dako, Glostrup, Denmark, S3006) (3 × 5 min each). Then, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min at RT and washed with washing buffer again (3 × 5 min each). DAKO protein block buffer (Dako, X0909) was added and incubated for 1 h at RT before incubating with primary antibodies specific for PGRMC1 (Abcam, ab48012), PHB1 (Abcam, ab75766), PHB2 (Cell signaling, 14084S) and Vimentin (Abcam, ab02547) overnight at 4 °C. The next day, cells were washed with washing buffer (3 × 5 min each) and incubated with secondary antibodies (Donkey-anti-goat, Alexa 488: Invitrogen, A11055; Donkey-anti-rabbit, Alexa 488: Invitrogen, A31573) for 1 h at RT in a humidified chamber in the dark. Nucleic acid was stained with DAPI (Thermo Fisher Scientific, 15733122) simultaneously with co-incubated secondary antibodies. After the final wash, the cells were mounted with Fluorescent Mounting Medium (Dako, S3023). Negative controls were prepared for each sample following the same staining procedure with isotype controls instead of primary antibodies. Fluorescence signals were detected with an Axioplan 2 Imaging fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

Proximity ligation assay

The in-situ proximity ligation assay (PLA) procedure was performed with the Duolink® PLA Kit (Sigma-Aldrich, DUO92008) and following the manufacturers protocol. The cells were incubated with the primary antibodies i.e., anti-PGRMC1 (Abcam, ab48012) with PHB1 (Abcam, ab75766) and PHB2 (Cell signaling, 14085S) overnight at 4 °C. The slides were washed twice for 5 min with buffer A, followed by incubation with the PLA probes (anti-goat PLUS and anti-rabbit MINUS) in antibody diluent for 60 min at 37 °C. After washing twice for 5 min with buffer A, ligation was performed using ligase diluted in ligation buffer for 30 min at 37 °C. Then the cells were washed with buffer A before incubation for 100 min with amplification stock solution at 37 °C. After washing twice for 10 min with buffer B, nuclear DNA was labeled with DAPI for 10 min and slides were mounted with mounting medium. Negative PLA control was performed using respective isotype control antibodies (isotype goat, Abcam, ab37373; isotype rabbit, Abcam, ab37415). Red fluorescence dots inside the cellular areas representing a single protein–protein interaction were quantified using image J software.

Western blotting

Cell suspensions were washed twice with ice cold PBS (Thermo Fisher Scientific, 2176323) and lysed in RIPA lysis buffer (50 mM TRIS (Sigma-Aldrich, 74,385), 150 mM NaCl (VWR corporation, 16C030032), 1% NP-40 (Sigma-Aldrich, 74,385), 0.5% Sodium deoxycholate (Sigma-Aldrich, D6750), 0.1% SDS (Sigma-Aldrich, S34121136), containing protease inhibitor (Roche, 49,422,800) and phosphatase inhibitor (Roche, 49121300). Protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). An amount of 20 µg of total protein was supplemented with 4 × Laemmli buffer (Bio-Rad, Feldkirchen, Germany, 1610747) containing 2-Mercaptoethanol (Sigma-Aldrich, M6250) and loaded onto Mini-PROTEAN® Precast Gels (Bio-Rad, 4568123) and separated via SDS-PAGE in SDS buffer (25 mM TRIS, 192 mM glycine (Sigma-Aldrich, 50046), 0.1% SDS, pH 8.3) at 100-150 V. Protein was transferred to Immun-Blot® PVDF Membranes (Bio-Rad,1620177) overnight at 4 °C and 10 mA in blotting buffer (20 mM TRIS, 200 mM glycine, 20% (v/v) methanol). Unspecific binding was blocked by incubation of the PVDF membrane with 5% skim milk powder (Sigma-Aldrich, 70166) in TRIS-buffer (20 mM TRIS, 150 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBS-T) for 1 h at RT. Primary antibodies including PGRMC1 (Cell signaling, Danvers, MA, USA, D6M5M), PHB1 (Cell signaling, 2426S), PHB2 (Cell signaling, 14085S) and ß-actin (Santa Cruz Biotechnology, sc-4778) were added in 5% skim milk—TBS-T and incubated overnight at 4 oC. Secondary antibodies were applied in 5% skim milk—TBS-T at RT for 1 h. Proteins were detected using Amersham™ ECL™ Western Blotting Detection Reagent (Cytiva, 17190731).

Subcellular protein fractionation

A subcellular protein fractionation kit (Thermo Fisher Scientific) was used to fractionate proteins into cytoplasmic, membrane, and nuclear fractions. Cells were harvested as pellets. The pellet was lysed with cytoplasmic extraction buffer, membrane extraction buffer, and nuclear extraction buffer. Primary antibodies specific for β-actin (Santa Cruz Biotechnology), Calreticulin (Santa Cruz Biotechnology), and Histon H3 (Cell signaling) were used to indicate the purity of the cytoplasmic, membrane, and nuclear fractions, respectively.

Co-immunoprecipitation

Co-immunoprecipitation was performed using the Pierce Co-IP kit (Thermo Fisher Scientific). Briefly, the anti-PGRMC1 antibody (Cell signaling) was first immobilized for 2 h using AminoLink Plus coupling resin. In parallel, cell pellets were resuspended in ice-cold IP Lysis buffer. An amount of 500 µg protein was incubated with resin at 4 °C overnight. After incubation, the resin was washed, and protein complexes bound to the antibody were eluted using elution buffer. Subsequent western blot analyses were performed as described before.

Gene silencing (siRNA Transfection)

To knock down PGRMC1 expression in T-HESCs, FlexiTube GeneSolution (Qiagen) was used, containing four siRNA(s) that specifically target human PGRMC1 mRNAs. Cells were transfected with the final concentration of 10 nM PGRMC1 siRNA(s) or negative control siRNA (siCTL) (Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to recommended procedures. Afterwards, cells were treated with decidualization medium containing either induction cocktail or DMSO, and harvested at different time points for downstream experiments. For PHB1 and PHB2 mRNA expression inhibition (siPHB1, siPHB2: Qiagen), the same siRNAs concentration was used.

Quantitative reverse-transcription PCR (qRT-PCR)

RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s specifications. Reverse transcription of RNA into cDNA was performed with the Omniscript RT kit (Qiagen) according to the manufacturer’s instructions. Quantitative PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen) and LightCycler ®480 System (Roche). Primers for PGRMC1 (Qiagen), PRL (Qiagen) and HPRT1 (Qrigene, Rockville, MD, USA). The delta-delta cycle threshold method was used to normalized expression to the reference gene HPRT1 [23, 24].

Statistical analysis

A two-tailed paired Student’s t-test was used to analyze experiments comparing two experimental groups or two-way ANOVA for multiple comparisons of more than two groups. A value of p < 0.05 was considered significant. All statistical analyses were performed with GraphPad Prism 9.0. Results were reported as means with standard deviation.