Figure 6. ERα is spatially associated... | Rockefeller University Press

Figure 6.

$Figure 6. ERα is spatially associated with β1-integrin and they are endocytosed together. (a) Widefield (top) and TIRFM (bottom) images of a coimmunofluorescence in MCF7 cells, using antibodies against β1-integrin (live-stained) and ERα. In the inset, white arrowheads indicate points of colocalization. Pearson’s correlation maps corresponding with the white box shown on the right. White arrowheads indicate points of positive Pearson’s correlation. (b) Quantification of a. Top left: Polar transformation of TIRFM images was performed using Fiji to align areas of the cell periphery where colocalization is found. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null), using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: nnull = 9 fields, nROI = 9 fields; Manders’: nnull = 14 fields, nROI = 9 fields). (c) Western blot of a coimmunoprecipitation in MCF7 cells, using antibodies against β1-integrin or ERα. Blotting antibodies are shown on the right. Input, whole lysate. IP, immunoprecipitated fraction. (d) ClustalW alignment of the eight β-integrins present in humans. The sequence of SRC1 is shown on top. NR-box motif is indicated in red. On the sequences of β1-integrin and β3-integrin, underlined in black is the region corresponding with their transmembrane domain, and in green is their cytoplasmic domain. The topology was predicted using the algorithm TMpred from the website ExPASy and the algorithm from the website TOPCONS. (e) Cartoon showing β1-integrin structure and putative interaction site with ERα. Black box indicates the localization of NR-box motif (LXXLL) within β1-integrin transmembrane domain. Red dot shows ERα palmitoylation site, and the arrow indicates where its helix 12 would be localized within the AF-2 domain. (f and g) Top: Western blot of total lysates of MCF7 cells, seeded on BSA (f) or FN (g) and treated for 60 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, shown is the β1-integrin/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test (n = 3 replicates). (h) Confocal images of MCF7 cells treated for 15 min as indicated and stained for β1-integrin (live stained) and ERα. Arrows indicate points of colocalization. Corresponding Pearson’s correlation maps are shown on the right, respectively. White arrows indicate points of positive Pearson’s correlation. (i) Quantification of h. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null) using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: nnull = 14 fields, nROI = 15 fields; Manders’: nnull = 10 fields, nROI = 11 fields). (j) Confocal images of MCF7 cells seeded on BSA or FN treated with E2 for 15 min and stained for β1-integrin and Rab11. Full images are shown in the insets. (k) Quantification of j. For each experimental condition, Pearson’s correlation index was calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by a one-tailed Student’s t test (per replicate: nBSA = 4 fields, nFN = 4 fields). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Shown data are representative of at least three independent experiments. Black arrowheads indicate positions of 100-kD markers. White arrowheads indicate positions of 50-kD markers. Treatments: ethanol (vehicle) or 10−8 M E2. Bars, 10 µm (unless otherwise indicated).$

ERα is spatially associated with β1-integrin and they are endocytosed together. (a) Widefield (top) and TIRFM (bottom) images of a coimmunofluorescence in MCF7 cells, using antibodies against β1-integrin (live-stained) and ERα. In the inset, white arrowheads indicate points of colocalization. Pearson’s correlation maps corresponding with the white box shown on the right. White arrowheads indicate points of positive Pearson’s correlation. (b) Quantification of a. Top left: Polar transformation of TIRFM images was performed using Fiji to align areas of the cell periphery where colocalization is found. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null), using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n_null = 9 fields, n_ROI = 9 fields; Manders’: n_null = 14 fields, n_ROI = 9 fields). (c) Western blot of a coimmunoprecipitation in MCF7 cells, using antibodies against β1-integrin or ERα. Blotting antibodies are shown on the right. Input, whole lysate. IP, immunoprecipitated fraction. (d) ClustalW alignment of the eight β-integrins present in humans. The sequence of SRC1 is shown on top. NR-box motif is indicated in red. On the sequences of β1-integrin and β3-integrin, underlined in black is the region corresponding with their transmembrane domain, and in green is their cytoplasmic domain. The topology was predicted using the algorithm TMpred from the website ExPASy and the algorithm from the website TOPCONS. (e) Cartoon showing β1-integrin structure and putative interaction site with ERα. Black box indicates the localization of NR-box motif (LXXLL) within β1-integrin transmembrane domain. Red dot shows ERα palmitoylation site, and the arrow indicates where its helix 12 would be localized within the AF-2 domain. (f and g) Top: Western blot of total lysates of MCF7 cells, seeded on BSA (f) or FN (g) and treated for 60 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, shown is the β1-integrin/β-actin density ratio normalized to the mean control group. Each symbol represents a different experiment. Differences between groups were analyzed by one-tailed paired Student’s t test (n = 3 replicates). (h) Confocal images of MCF7 cells treated for 15 min as indicated and stained for β1-integrin (live stained) and ERα. Arrows indicate points of colocalization. Corresponding Pearson’s correlation maps are shown on the right, respectively. White arrows indicate points of positive Pearson’s correlation. (i) Quantification of h. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization (ROI) and compared with random areas without colocalization (Null) using Fiji. For Pearson’s correlation, datasets are plotted and mean ± SD are shown on the graph. For Manders’ coefficients, the table shows mean and SD for each dataset. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n_null = 14 fields, n_ROI = 15 fields; Manders’: n_null = 10 fields, n_ROI = 11 fields). (j) Confocal images of MCF7 cells seeded on BSA or FN treated with E₂ for 15 min and stained for β1-integrin and Rab11. Full images are shown in the insets. (k) Quantification of j. For each experimental condition, Pearson’s correlation index was calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by a one-tailed Student’s t test (per replicate: n_BSA = 4 fields, n_FN = 4 fields). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Shown data are representative of at least three independent experiments. Black arrowheads indicate positions of 100-kD markers. White arrowheads indicate positions of 50-kD markers. Treatments: ethanol (vehicle) or 10⁻⁸ M E₂. Bars, 10 µm (unless otherwise indicated).