Figure 5. ERα is localized in Rab11... | Rockefeller University Press

Figure 5.

$Figure 5. ERα+ is localized in Rab11+ vesicles in the presence of FN. (a) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min as indicated and stained for ERα. Panels show the cytoplasmic/plasma membrane (basal) plane (z2). Nuclear/cytoplasmic (apical) plane (z1) is shown in Fig. S3 b. White arrows indicate ERα+ vesicles determined as punctae of 10–15 pixels in diameter (∼200 nm). (b) Quantification of a. Apical (nuclear/cytoplasmic) versus basal (cytoplasmic/plasma membrane) distribution of ERα+ vesicles. Structures of 10–15 pixels in diameter were quantified using Fiji. Mean number of endosomes in each fraction and for each condition is shown. (c) Heatmaps of T47D cells seeded on BSA or FN treated for 60 min as indicated and stained for ERα. Cells are outlined in white. Dashed line outlines the nucleus. Intensity bars are shown on the right (red, maximum pixel intensity; blue, minimum pixel intensity). Original images are shown in the insets. (d) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 60 min with E2 and stained for ERα and Rab11. Arrows indicate areas of colocalization within filopodia. Pearson’s colocalization maps are shown in the insets. (e) Quantification of d. For each experimental condition, Pearson’s correlation index was calculated within filopodia protrusions using Fiji. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: nBSA = 16 fields, nFN = 15 fields). (f) Quantification of d. For each experimental condition, overall Rab11 intensity was calculated using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: nBSA = 5 fields, nFN = 4 fields). (g) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min with E2 in the presence of dextran-CF543 and stained for Rab11. Arrows indicate areas of colocalization between the two fluorophores. Higher magnification is shown in the inset. ***, P < 0.001. Shown data are representative of at least three independent experiments. Treatments: ethanol (vehicle) or 10−8 M E2. Bars, 10 µm (unless otherwise indicated).$

ERα⁺ is localized in Rab11⁺ vesicles in the presence of FN. (a) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min as indicated and stained for ERα. Panels show the cytoplasmic/plasma membrane (basal) plane (z2). Nuclear/cytoplasmic (apical) plane (z1) is shown in Fig. S3 b. White arrows indicate ERα⁺ vesicles determined as punctae of 10–15 pixels in diameter (∼200 nm). (b) Quantification of a. Apical (nuclear/cytoplasmic) versus basal (cytoplasmic/plasma membrane) distribution of ERα⁺ vesicles. Structures of 10–15 pixels in diameter were quantified using Fiji. Mean number of endosomes in each fraction and for each condition is shown. (c) Heatmaps of T47D cells seeded on BSA or FN treated for 60 min as indicated and stained for ERα. Cells are outlined in white. Dashed line outlines the nucleus. Intensity bars are shown on the right (red, maximum pixel intensity; blue, minimum pixel intensity). Original images are shown in the insets. (d) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 60 min with E₂ and stained for ERα and Rab11. Arrows indicate areas of colocalization within filopodia. Pearson’s colocalization maps are shown in the insets. (e) Quantification of d. For each experimental condition, Pearson’s correlation index was calculated within filopodia protrusions using Fiji. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n_BSA = 16 fields, n_FN = 15 fields). (f) Quantification of d. For each experimental condition, overall Rab11 intensity was calculated using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n_BSA = 5 fields, n_FN = 4 fields). (g) Images of confocal microscopy of MCF7 cells seeded on BSA or FN treated for 15 min with E₂ in the presence of dextran-CF543 and stained for Rab11. Arrows indicate areas of colocalization between the two fluorophores. Higher magnification is shown in the inset. ***, P < 0.001. Shown data are representative of at least three independent experiments. Treatments: ethanol (vehicle) or 10⁻⁸ M E₂. Bars, 10 µm (unless otherwise indicated).