Fig 2: GPER localized to the nucleus is unable to stimulate MAPK signaling, but can be recruited to the promoters of its target genes c-FOS and CTGFA. Immunoblots showing ERK1/2 phosphorylation upon activation of GPER with OHT. Other panels display the total levels of ERK2, and the levels of endogenous GPER and exogenous FLAG-GPER in transfected SkBr3 cells. The transfected SkBr3 cells were treated for 30 min with vehicle (-) or 10 µM OHT. The panel on the right shows the densitometric quantitation of the blots normalized to ERK2 expression levels; each bar represents the average of two independent experiments with the lines on top indicating the range of the two values; the black dots indicate the samples, arbitrarily set to 100%, relative to which the values of the other samples of a set were calculated. B. GPER is recruited to the promoters of its target genes c-FOS and CTGF in CAFs_I. CAFs_I and SkBr3 cells were used to perform a ChIP assay with an anti-GPER antibody. C. Tunicamycin treatment induces the recruitment of GPER on the promoters of its target genes in SkBr3 cells, treated for 24 hours with 5 µg/ml tunicamycin and then used to perform ChIP experiments using an anti-GPER antibody. D. ChIP experiment to assess recruitment of GPER to target sites with SkBr3 cells transfected for 24 hours with shRNA or shGPER constructs and then treated for additional 24 hours with 5 µg/ml tunicamycin. ChIP experiments were performed using an anti-GPER antibody. E. Control RT-PCR experiment to verify the effectiveness of shGPER-mediated knock-down of the GPER mRNA 48 hours after transfection. Values were normalized to GAPDH expression, and presented as fold change (mean ± SD) of shGPER transfected cells relative to cells transfected with a control shRNA construct. F. ChIP experiment to assess recruitment of NLS-defective GPER to target sites. In this case, the ChIP was done with an anti-FLAG antibody. In ChIP experiments, the specific precipitation of AT-rich sequences from the c-FOS and CTGF promoters was evaluated by real-time PCR; enrichment was calculated relative to input (in panel B) as well as standardized to the untreated controls set to 1 (in panels C, D, and F). Bar graphs show the means ± SD of three independent experiments. *, p-value = 0.05 for comparison to respective untreated (or shRNA) control; §, p-value = 0.05 for comparison to corresponding wild-type GPER plasmid (panel F).

Fig 3: Inhibition of N-linked glycosylation induces the nuclear accumulation of GPERA. Schematic representation of the GPER protein structure. The asparagine residues N25, N32 and N44 are predicted N-linked glycosylation sites in the N-terminal portion of the receptor. B. Representative immunofluorescence micrographs of endogenous GPER in SkBr3 breast cancer cells treated or not for 24 hours with 5 µg/ml of tunicamycin and stained with an anti-GPER antibody (green staining). Nuclei were stained with DAPI (blue). Images shown are representative of 10 different random fields. C. GPER immunoflourescence micrographs of SkBr3 cells transfected for 24 hours with shRNA or shGPER constructs, or co-transfected, for additional 24 hours, with shGPER in combination with the shRNA-resistant GPER expression vector for either the wild-type GPER (GPER Rescue) or the N-glycosylation double mutant N25/32Q. Images shown are representative of 10 different random fields. D. Immunoblot of a subcellular fractionation experiment with SkBr3 breast cancer cells transfected as indicated. For these experiments, exogenous GPER is FLAG-tagged. Tubulin and histone H3 serve as markers and for standardization for the cytoplasmic and nuclear compartments, respectively. The bottom panel shows the densitometric analysis with each data point representing the mean ± SD of three independent experiments. After standardization to the respective compartmental markers, the values were expressed as % of the respective samples with FLAG-tagged wild-type GPER (each set to 100%).

Fig 4: GPER localizes to both in the cytoplasm and the nucleus of CAFs A. and normal fibroblasts B. from biopsies of in-situ breast carcinomaSamples are from patients #6-8. C. Immunofluorescent staining of CAFs_I and epithelial cells from an unrelated breast carcinoma sample. Samples were stained with an anti-GPER antibody (green) and DAPI (blue), and analyzed by confocal microscopy. Immunofluorescence micrographs are representative of 10 different random fields.

Fig 5: CAFs and normal fibroblasts from a panel of breast cancer biopsies are all heterozygous for the P16L polymorphismDirect amplicon sequencing of the GPER1 portion encoding the N-terminus of GPER of CAFs and normal fibroblasts from patients #1-5 with invasive carcinoma A. and from patients #6-8 with in-situ carcinoma and independent normal epithelial cells for comparison B. Note that all fibroblasts in this set of samples display a double peak with both C and T at the relevant position (arrows). C. Scheme of the analytical PCR protocol to confirm the presence of the C/T polymorphism indicated by sequencing. Using two common outer primers (Outer Fw and Outer Rv) and two allele-specific inner primers (Inner-C and Inner-T), PCR products of specific diagnostic sizes are generated. D. Image of an agarose gel for the genotyping of all samples with the PCR scheme of panel C. T and N stand for tumor (CAFs) and normal fibroblasts, respectively. Note that fibroblasts of patients #1-8 are heterozygous, CAFs_I homozygous for the variant with the T allele, and eptihelial cells from an unrelated breast carcinoma sample and SkBr3 cells are homozygous for the C allele.