Finch AR , Sedgley KR , Caunt CJ , McArdle CA .

062918 Wellcome Trust , 076557 Wellcome Trust , 078407 Wellcome Trust , Wellcome Trust

	Figure 1. Relationship between Ad titer and receptor expression in Ad GnRHR-infected MCF7 cells. MCF7 cells infected with the indicated titers of Ad expressing N-terminal HA-tagged hGnRHRs, h.XGnRHRs, or XGnRHRs were stained for cell surface receptors (incubation of intact cells with primary antibody) and nuclei (DAPI) or for whole cell receptor expression (permeabilization before primary antibody addition), prior to image acquisition and analysis as described in the Materials and Methods. The upper figures show stain intensity (arbitrary fluorescence units, AFU) at the cell surface (open symbols) or throughout the cell (whole cell, filled symbols) in the positive cells in a single representative experiment (mean±s.e.m., n=4). The lower panels show representative images of HA-GnRHR staining at the cell surface or throughout the whole cell and are from cells infected with Ad hGnRHR at 300 pfu/nl, and Ad h.XGnRHR or Ad XGnRHR each at 100 pfu/nl (as indicated). These images show <1% of the total area imaged to generate the graphs shown. Two-way ANOVA revealed that Ad titer and permeabilization were statistically significant variables (P<0·01) for each receptor type.
	Figure 2. GnRHR localization at matched whole cell expression levels. MCF7 cells were treated as described for Fig. 1 except that they were incubated for 16 h with 0 (open bars) or 1·8×10−7 M IN3 (filled bars) before staining for HA-tagged receptors in intact (cell surface) and permeabilized (whole cell) cells as indicated. The figure shows the proportion of cells with cell surface staining (%+ve, panel A), the cell surface staining in those cells (AFU, panel B), and the expression index calculated by compounding these values (panel C). Fluorescence intensity and expression index were also determined for whole cell staining (panels D and E respectively) and used for calculation of the proportion of receptors at the cell surface (proportional cell surface expression, PCSE, panel F). These data are from the experiment shown in Fig. 1 (mean±s.e.m., n=4) with Ad titers selected for comparable whole cell expression levels (300 pfu/nl for Ad hGnRHR and 100 pfu/nl for Ad h.XGnRHR). Parallel experiments with the XGnRHR (100 pfu/nl) yielded PCSE values of 39·1±5·9 and 59·1±25·6 in control and IN3-treated cells respectively. Two-way ANOVAs of the graphed data revealed IN3 as a significant variable for each measure except for AFU in whole cells (e.g., P<0·05 for panels A–C, and F, but not for panel D). The lower panel images show representative views of DAPI-stained nuclei and HA-GnRHR expressing cells as well as merged images. Segmentation is also shown (lower right) to illustrate how the individual cells were filtered to identify HA-GnRH positive cells (green outlines, AFU >10% above background) and negative cells (red outlines, AFU <10% above background). Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-07-0471. The horizontal scale bar (DAPI image) is ∼20 μm.
	Figure 3. Time dependence of the IN3 effect on plasma membrane expression of HA-GnRHRs. MCF7 cells infected with Ad HA-hGnRHRs (upper panels) or Ad HA-h.XGnRHRs (lower panels), incubated for the indicated time with 0 (open circles) or 1·8×10−7 M (filled circles) IN3 prior to staining and imaging as described under Fig. 2. The figure shows expression indices (left panels) and the PCSE values (right panels) pooled from four separate experiments (mean±s.e.m., n=4) each with quadruplicate observations. Two-way ANOVA revealed time and IN3 as significant variables for each panel (P<0·01). IN3 effects were statistically significant (*P<0·05, **P<0·01 using Student's t-test) compared with time-matched controls, as indicated.
	Figure 4. Concentration dependence of the IN3 effect on plasma membrane expression of HA-GnRHRs. MCF7 cells were transduced with Ad HA-GnRHRs and incubated for 16 h with the indicated concentration of IN3 prior to staining and imaging. Data shown are cell surface expression indices from a representative experiment. Two-way ANOVA revealed receptor type and concentration as significant variables (P<0·01) and IN3 significantly increased cell surface GnRHR expression as indicated (*P<0·05, **P<0·01 by Student's t-test).
	Figure 5. Effects of IN3 on plasma membrane expression of HA-GnRHRs in MCF7 and DU145 cells. MCF7 (upper panels) and DU145 (lower panels) cells infected with Ad expressing the indicated GnRHRs and incubated for 16 h in medium with 0 (ctrl., open bars) or 1·8×10−7 M IN3 (filled bars) before imaging of cell surface and whole cell receptors. The left panels show expression indices and the right panels show PCSE values, pooled from four separate experiments each with three or four replicates (mean±s.e.m., n=4). Two-way ANOVAs revealed IN3 as a significant variable (P<0·05) for each receptor and each endpoint, and the effects of IN3 on PCSE were statistically significant (*P<0·05, **P<0·01 when compared with control values matched for cell and receptor type) for both receptors and both cell types.
	Figure 6. Effects of IN3 on plasma membrane expression of HA-GnRHRs in MCF7 and LβT2 cells. LβT2 cells were cultured in plates that had been pretreated with Matrigel and MCF7 cells were either on Matrigel-treated plates (lower panel) or untreated plates (middle panel). The cells were infected with Ad expressing HA-tagged GnRHRs and then incubated for 16 h in medium with 0 (control) or 1·8×10−7 M IN3, as indicated, before imaging. The figure shows PCSE values pooled from seven separate experiments each with four replicates (mean±s.e.m., n=4–7). ANOVAs revealed IN3 as a significant variable (P<0·01) and the effects of IN3 were statistically significant (*P<0·05, **P<0·01 when compared with control values for matched cell and receptor type) for both receptors and both cell types.
	Figure 7. Effect of IN3 on GnRHR-mediated [3H]IP responses. MCF7 cells were infected with Ad expressing hGnRHR, h.XGnRHR, or mGnRHR and then transferred to media with 0 or 1·8×10−7 M IN3 and [3H]inositol (for 16 h), then washed and stimulated for 1 h with the indicated concentration of Buserelin with 10 mM LiCl and either 0 (control) or 1·8×10−7 M IN3. Panels A–C show data for cells receiving no IN3 (control), as well as for cells receiving IN3 during the stimulation with Buserelin (co-incubation) or during both the 16-h pretreatment and the Buserelin stimulation (pre- and co-incubation) and these data are normalized as a percentage of the maximal response to Buserelin in control cells (those receiving no IN3). These data are pooled from three separate experiments each with triplicate observations (mean±s.e.m., n=2–3) and the control values were 592±52, 520±101, and 1337±91 c.p.m./well (n=3) for the hGnRHR, h.XGnRHR, and mGnRHR respectively. Panel D shows the increment in the response due to the IN3 pre-incubation (e.g., the response in cells pre-treated and co-incubated with IN3 minus the response in cell receiving IN3 co-incubation alone) for all three receptors, and the data in panel D are calculated from those in panels A–C. Maximal hGnRHR-mediated [3H]IP accumulation was significantly increased by pre- and co-incubation with IN3 (* P<0·05, panel A), whereas it caused no such increase in the maximal h.XGnRHR-mediated or mGnRHR-mediated responses (panels B and C). The effect of pre-incubation was statistically significant for 10−10–10−6 M Buserelin acting via the hGnRHR (**P<0·01, *P<0·05; panel D) but not for any concentration of Buserelin acting via the h.XGnRHR or mGnRHR (P>0·1, panel D).
	Figure 8. Effects of Buserelin and IN3 on cell number and nuclear stains. Control and Ad hGnRHR infected cells were cultured in medium with 2% FCS with or without Buserelin (10−7 M) and IN3 (1·8×10−7 M) as indicated, prior to staining and imaging to determine cell number (in the imaged area of 2·4 mm2) as well as the proportion of sub-2n and post-mitotic cells. The bar charts are from a single representative experiment (mean±s.e.m., n=4, *P<0·05, **P<0·01 by Student's t-test). Analysis of data pooled from three similar experiments by two-way ANOVAs revealed that Buserelin was not a significant variable in control cells but was in Ad hGnRHR-infected cells (P<0·01), and in Ad hGnRHR-infected cells the reduction of cell number was greater in cells treated with IN3 and Buserelin than in cells receiving Buserelin alone (P<0·05 by Student's t-test). The inset in panel B shows a distribution profile with integrated nuclear stain intensity on the horizontal axis (arbitrary fluorescence units) and cell number (in thousands) on the vertical axis, demonstrating the 2n and 4n cell populations and the arbitrary cut-off (<50% of the 2n peak) used to define sub-2n cells. Panel D shows a small proportion of the image captured from a single well, illustrating the small and highly stained nuclei that were characteristically seen in pairs and identified by the analysis algorithm as post-mitotic (anaphase) cells (arrows). Horizontal scale bar=∼20 μm.
	Figure 9. Effects of membrane-permeant and membrane-impermeant ligands on plasma membrane expression of HA-GnRHRs. MCF7 cells were infected with Ad expressing HA-hGnRHRs (upper panel) or h.XGnRHRs (lower panel) and then incubated for 16 h with control medium (ctrl.) or medium supplemented with IN3 (1·8×10−6 M), antide (ant.; 10−8 M), cetrorelix (cet.; 10−8 M), or GnRH (G, 10−8 M) before imaging. The data shown are PCSE values pooled from seven separate experiments each with three or four replicates (mean±s.e.m., n=4–7) and the effects of IN3 were statistically significant at both receptors, whereas the effects of antide and cetrorelix were significant only at the HA-h.XGnRHRs (*P<0·05, **P<0·01 compared with control values for receptor type).
	Figure 10. Time course of effects of membrane-permeant and membrane-impermeant ligands on plasma membrane expression of HA-h.XGnRHRs. Upper panel: MCF7 cells were cultured and treated as described under Fig. 9, except that only the HA-h.XGnRHR was used and incubation times were varied as indicated. The data shown are expression indices pooled from four separate experiments each with three replicates (mean±s.e.m., n=4). Two-way ANOVA revealed that treatment and time were both significant variables (P<0·01) and the effects of all antagonists were significant (P<0·05 compared with time-matched controls) at 24 and 48 h. The effects of IN3 (but not cetrorelix or antide) were also significant (P<0·05) after 1 and 2 h of incubation. Lower panels: MCF7 cells were cultured and transduced with Ad HA-GnRHR or Ad h.XGnRHR and then incubated 16–24 h with or without IN3 (1·8×10−6 M) and cetrorelix (cet., 10−8 M) before staining and imaging. The data shown are pooled from four separate experiments each with three replicates (mean±s.e.m., n=4). Two-way ANOVA revealed IN3 as a significant variable at both receptors and cetrorelix as a significant variable at the HA-h.XGnRHRs (P<0·01) but the IN3–cetrorelix interaction was not statistically significant at either receptor (P>0·05). *P<0·05, **P<0·01 compared with controls by Student's t-test.

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