PMC

(A) Real-time qPCR analysis to detect the relative expression levels of ERα (ESR1) and TFF1 prepared from MCF-7, MCF10A, LCC1 and LCC9 cells grown in phenol red containing DMEM. (B) Box plots showing inter-probe distances (d) between heterologous TFF1 and GREB1 alleles, as measured by 2D FISH in nuclei of LCC1 or LCC9 cells grown in the absence of E2 (-E2) or after 1 hr in the presence of 100 nM E2. Asterisks indicate data points beyond the 95^th percentile. N = 50 cells.

(A) 3D Interphase FISH with probes for TFF1 (red) and GREB1(green) on nuclei of MCF-7 cells grown either in the absence of estrogen (-E2), or 1 and 16 hrs after the addition of 10 nM (top row) or 100 nM (middle row) E2 (+E2). Bottom row: 1 h and 16 hrs of 100 nM E2 addition following pre-treament with 2.5 nM α-amanitin. Nuclei were stained with DAPI (blue). Scale bars  = 5 µm. (B) Box plots show inter-probe distances (µm) between homologous or heterologous TFF1 and GREB1 alleles, as measured either by 3D FISH in nuclei of MCF-7 cells grown in the absence of E2 (-E2) or 1 and 16 hr after the addition of 10 nM (top) or 100 nM (middle) E2. Bottom row: 1 h and 16 hrs of 100 nM E2 addition following pre-treament with 2.5 nM α–amanitin. Asterisks indicate data points beyond the 95^th percentile. N = 40 cells.

(A) Western blot to detect protein expression of ERα and GAPDH in total cell lysate prepared from HMEC, MCF10A and MCF-7 cells. (B) Immunohistochemical staining of HMEC and MCF-7 cells with antibody that detects ERα (brown). (C) FISH with probes for TFF1 and GREB1, and paints for chromosomes 2 and 21, on metaphase chromosomes prepared from MCF-7 cells. Bars, 10 µm. (D) Real-time qPCR analysis to detect the relative expression levels of GREB1, TFF1 and a RPLP0 control prepared from MCF-7 cells grown without E2 (-E2) and during 16 hr time courses in the presence of either 10 nM or 100 nM E2. Cells with (+) and without (−) pre-treatment with 2.5 nM α–amanitin were also examined.

(A) 3D interphase FISH with chromosome paint to chromosome territory 2 (red) and 21 (green) on nuclei from MCF10A cells imaged in the absence of estrogen (-E2), or after 60 mins of 10 nM E2 addition (+E2). Nuclei were stained with DAPI (blue). Scale bars  = 5 µm. (B) Histograms showing the percentage of chromosome paint hybridization signal normalized to the percentage of DAPI signal, before and after E2 addition, across 5 erosion shells placed between the edge (shell 1) and the centre (shell 5) of the nuclei. N = 25−30 cells. (C) 3D interphase FISH with chromosome paint to chromosome territory 2 (red) and 21 (green) on nuclei from MCF-7 cells imaged in the absence of estrogen (-E2), or after 60 mins of 100 nM E2 addition (+E2). Nuclei were stained with DAPI (blue). Scale bars  = 5 µm. (D) Box plots show the percentage of chromosome paint hybridization signal (CT2 and CT21) normalized to the percentage of DAPI signal, before and after E2 addition. Asterisks indicate data points beyond the 95^th percentile. N = 30 cells.

(A) FISH with probes for CTSD (blue) and PGR (green) and paint for chromosome 11 (red) on metaphase chromosome spreads of MCF-7 cells. Scale bars  = 10 µm. (B) Real-time qPCR analysis to detect the relative expression levels of PGR, CTSD and a RPLP0 control prepared from MCF-7 cells grown without E2 (-E2) and during 16 hr time courses in the presence of 10 nM E2. (C) 3D Interphase FISH with probes for CTSD(red), PGR(green) and the centromere of chromosome 11 CEP11 (blue) on nuclei of MCF-7 cells grown in the absence of estrogen (-E2) and 3 hrs after the addition of 10 nM E2 (+E2). Nuclei were counterstained with DAPI (blue). Scale bars  = 5 µm. (D) Box plots show inter- and intra-chromosomal distances (µm) between PGR and CTSD alleles, as measured by 3D FISH in nuclei of MCF-7 cells grown in the absence of E2 (-E2) and 3 hrs after the addition of 10 nM E2. Asterisks indicate data points beyond the 95^th percentile. N = 50 cells.

(A) Interphase FISH with probes (red or green) for TFF1 and GREB1 on nuclei from HMEC and MCF10A cells in the absence of estradiol (-E2), or after 60 mins of E2 addition (+E2). Nuclei were stained with DAPI (blue). Scale bars, 10 µm and 5 µm. (B) Box plots showing; top: inter-probe distances (d in µm) and bottom: inter-probe distances (d) normalized to nuclear radius (r) between homologous or heterologous TFF1 and GREB1 alleles, as measured either by 3D FISH in HMEC nuclei grown in the absence (-E2) or after 1 hr of 100 nM E2 addition. Shaded boxes show the mean and 25–75 percentiles of the data. Asterisks indicate data points beyond the 95^th percentile. N = 50 cells. (C) Histograms showing the percentage of signals of the radial position of TFF1 and GREB1 alleles, before and after E2 addition, across 5 erosion shells placed between the edge (shell 1) and the centre (shell 5) of the nuclei. N = 50 cells. (D) Box plots showing inter-probe distances (µm) between homologous or heterologous TFF1 and GREB1 alleles, as measured by 3D FISH in MCF10A nuclei grown in the absence (-E2) or after 1 hr of 10 nM E2 addition. Asterisks indicate data points beyond the 95^th percentile. N = 50 cells.