Fig 2: Phosphoproteome analysis connects PGRMC1 signalling to breast cancer survival pathways.a, b Volcano plots obtained from phosphoproteome analysis representing both total and phosphorylated protein forms following 50 μM AG-205 treatment or PGRMC1 silencing in ZR-75-1 and MDA-MB-468 cells. c Protein classes involved in the phosphoproteome analysis generated by PANTHER Protein Class subsets. d Gene ontology (GO) classification of biological processes involved in the phosphoproteome analysis generated by PANTHER GO-Slim Biological Process subsets. e, f Rows represent the top five GO term- enriched altered signalling pathways analysed by REACTOME and heatmap clusters of differentially expressed genes identified by phosphoproteome analysis following either 50 μM AG-205 treatment or PGRMC1 silencing. Pink represents pathways and genes associated with ZR-75-1, and orange represents pathways and genes associated with MDA-MB-468 cells. Volcano plots: significant down- and upregulated proteins are represented, P < 0.05.

Fig 3: Progesterone receptor membrane-associated component 1 (PGRMC1) is required for lipid accumulation during 3T3L1 cells differentiation.a Analyses of protein expressions in differentiated 3T3L1 cells (control, KD#1, or KD#2) by western blotting using antibodies against PGRMC1 or GAPDH. b Oil Red O staining of differentiated 3T3L1. Control or two types of stable PGRMC1-KD 3T3L1 cells (PGRMC1 KD#1 and KD#2) were differentiated and stained with Oil Red O. The microscope images are shown in the upper panels of (b). Graphs in the lower panel of (b) depict the absorbance at 490 nm by Oil Red O (n = 8). c Analyses of mRNA expression of PGRMC1, CEBPβ, PPARγ, or FABP4 in 3T3L1 cells at the indicated time periods during differentiation (control, PGRMC1 KD) by quantitative PCR (qPCR) (n = 10). Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. d Analyses of protein expressions in 3T3L1 control cells during differentiation by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. e Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (Control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data represent mean ± S.E. Statistical analyses were performed using ANOVA with Tukey’s T test. *P < 0.05. †P < 0.05 (vs control day 0 (c)). #P < 0.05 (PGRMC1 KD vs control on the same day (c)).

Fig 4: Progesterone receptor membrane-associated component 1 (PGRMC1) contributed to low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) uptake by regulating the translocation of LDL-R and VLDL-R.a, b Analyses of the effect to LDL uptake by PGRMC1. a Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with Alexa Fluor 488-acetylated LDL (green) and DAPI (blue) are shown (scale bar: 10 μm). b Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with Alexa Fluor 488-acetylated LDL for 60 min. The graph shows the mean of fluorescence intensities (per 10,000 cells) (n = 4). c, d Analyses of the effect to VLDL uptake by PGRMC1. c Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with DiI–VLDL (red) and DAPI (blue) are shown (scale bar: 10 μm). d Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with DiI–VLDL for 60 min. (n = 4). e Analyses of regulation of the LDL-R and VLDL-R localization by PGRMC1. Protein expressions in whole-cell lysates were detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, GAPDH, or PGRMC1. f After plasma membrane fractions of 3T3L1 cells (control and PGRMC1 KD) were extracted, protein expression in the plasma membrane was detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, Na-K ATPase α1, or PGRMC1. f, g Co-immunoprecipitation assay for interaction between PGRMC1 and LDL-R, or PGRMC1 and VLDL-R. FLAG-PGRMC1 WT or Y113F was overexpressed in 3T3L1 cells, and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1, anti-LDL-R antibody, or anti-VLDL-R antibody. h Co-immunoprecipitation assay for interaction between endogenous PGRMC1 and LDL-R. 3T3L1 cell lysate was incubated with anti-PGRMC1 antibody or normal rabbit IgG, and then incubated with 10 µl protein A-sepharose beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-LDL-R antibody. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test (b, d). *P < 0.05.

Fig 5: Progesterone receptor membrane-associated component 1 (PGRMC1) contributes to glucose uptake by regulating the translocation to the plasma membrane of GLUT4.a Analysis of the effect of insulin-stimulated 2-deoxyglucose (DG) uptake by PGRMC1. After treatment with or without 0.5 μmol l−1 insulin for 18 min, 3T3L1 cells (control or PGRMC1 KD) were incubated with 1 μmol l−1 2-DG for 20 min, and the 2-DG uptake was measured. The graph shows relative fold change by normalizing with 2-DG uptake of 3T3L1 control cells without treatment of insulin (n = 4). b Analysis of the effect of fatty acid synthesis by PGRMC1 using [13C6]-glucose. After differentiated 3T3L1 cells (control and KD) were incubated with 4.5 g l−1 [13C6]-glucose for 24 h, the fatty acids were extracted. [13C6]-glucose, [13C4–12]-palmitic acid, [13C4–16]-stearic acid, and [13C4–16]-oleic acid in cells were measured by LC/MS. (n = 3). See Supplementary Fig. 7 for more detail. c Analyses of regulation of the GLUT4 translocation by PGRMC1. Plasma membrane fractions were extracted from 3T3L1 cells (control and PGRMC1 KD) treated with or without insulin. The expressed proteins in the plasma membrane or whole-cell lysate were detected by western blotting using antibodies against GLUT4, GLUT1, PGRMC1, phosphorylated Akt (pAkt), Akt, Na-K ATPase α1, or GAPDH. d Co-immunoprecipitation assay for interaction between PGRMC1 and GLUT4. FLAG-PGRMC1 WT or Y113F was overexpressed in 3T3L1 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-GLUT4 antibody. e Co-immunoprecipitation assay for interaction between endogenous PGRMC1 and GLUT4. 3T3L1 cell lysate was incubated with anti-PGRMC1 antibody or normal rabbit IgG, and then incubated with 10 µl of protein A-sepharose beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-GLUT4 antibody. All data are represented as mean ± S.E. Statistical analysis was performed using Student’s T test. *P < 0.05.