DOI: 10.6084/m9.figshare.25137995
title: Additional file 1 of Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization

description: Additional file 1: Supplementary Fig. 1. Rise-to-decline expression pattern of PGRMC1 is linked to the decidualization program. (A) PGRMC1 protein expression changes during 9 days of decidualization were measured by western blot in the St-T1 cell line. (B) PGRMC1 protein expression changes during 10 days of stimulation with MPA, cAMP, and DMSO, respectively, were measured by western blot in T-HESCs. (C) PGRMC1 protein expression levels on day 6 and day 10 when cultured with DMSO, nomegestrel (NOM), P4, cAMP, MPA/cAMP (M + A), and MPA, respectively, measured by western blot in T-HESCs. Supplementary Fig. 2. PGRMC1 is effectively downregulated by siRNA on protein level. (A) The PGRMC1 protein expression on day 2 and day 10 after transfection of T-HESCs with either 10 nM anti-PGRMC1 siRNA (siPGRMC1) or unspecific scrambled-control siRNA (siCTL). (B) A comparison of the PGRMC1 protein expression changes within 10 days after transfection of T-HESCs with either 10 nM siPGRMC1 or 10 nM siCTL. Supplementary Fig. 3. PGRMC1-downregulation before decidualization induction impairs morphological remodeling of T-HESCs. The cellular morphology changes of the T-HESCs induced with either DMSO (upper panel) or MPA/cAMP (down panel) after 10 days of siRNA treatment (siCTL, left panel; siPGRMC1, right panel). Scale bar: 200 µm. Supplementary Fig. 4. PGRMC1-downregulation after decidualization induction does not impair morphological remodeling of T-HESCs. The cellular morphology changes of the T-HESCs induced with either DMSO (non-induction, column 1) or MPA/cAMP (Induction, columns 2–4). I (column 3) and II (column 4) indicate that siRNA treatment on T-HESCs was conducted on day 2 or day 4 of decidualization induction, respectively. Scale bar: 200 µm. Supplementary Fig. 5. PGRMC1-downregulation after progestin induction does not impair decidualization in the St-T1 cell line. The mRNA expression levels of PGRMC1 (A, C) and PRL (B, D) in St-T1 treated with MPA/cAMP for decidualization induction (red line), and non-induction (black line). The mRNA expression levels of PGRMC1 and PRL in St-T1 cells transfected with 10 nM siPGRMC1 (blue line) on the second (A, B) and fourth day (C, D) after decidualization induction, respectively. Results are shown as the mean ± SEM from three independent biological replicates. Statistical analysis was performed by a two-way ANOVA test. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. The red * indicates the comparison between the red and black lines. The blue * indicates the comparison between the red and blue lines. Supplementary Fig. 6. PGRMC1 and PHB1/PHB2 co-localize in T-HESCs. Double Immunofluorescence staining for PGRMC1 (red) and PHB1 (green) or PHB2 (green) in T-HESCs treated with DMSO (A) as control or MPA/cAMP (B) for decidualization induction. Magnification: 40x. Scale bar: 20 µm. Supplementary Fig. 7. PGRMC1 does not interact with PHBs without induction. The interactions between PGRMC1 and PHB1 (A) and PHB2 (B) in T-HESCs without induction were analyzed with proximity ligation assay from day 2 to day 10. Each red spot represents a single interaction. Nuclear stain: DAPI. Magnification 40X. Supplementary Fig. 8. PGRMC1 co-precipitate with PHB1/PHB2 upon decidualization induction. PGRMC1 was immunopurified from native whole cell lysates of T-HESCs using anti-PGRMC1 antibody. Western blot analyses of co-immunoprecipitated PHB1 (upper) and PHB2 (bottom) in T-HESCs with and without decidualization induction. Supplementary Fig. 9. Morphological changes during decidualization upon PHBs downregulation. The cellular morphology changes of the T-HESCs induced with either DMSO (non-Induction) or MPA/cAMP (Induction) upon either PHBs knockdown alone or both. Scale bar: 200 µm. Supplementary Fig. 10. Overview over the role of PGRMC1 in human endometrial decidualization. Upon stimulation with progesterone or MPA, the PGRMC1 rise-to-decline changes are essential for successful decidualization of the human endometrial cells (upper panel). With downregulated PGRMC1 expression before induction, the decidualization program cannot be carried out, leading to decidualization failure (bottom panel).