Figure 3. E 2 stimulates endocytosis of... | Rockefeller University Press

Figure 3.

$Figure 3. E2 stimulates endocytosis of vesicles containing ERα. (a) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for ERα. Arrows indicate ERα+ endosomes. (b) Quantification of a. For each experimental condition, structures of ∼200-nm diameter (10–15 pixels) were quantified using Fiji. Shown is the number of ERα+ puncta per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: nvehicle = 81 cells, nE2 = 87 cells; FN: nvehicle = 50 cells, nE2 = 64 cells). (c) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for EEA1. Cells are delineated in white. Arrows indicate early endosomes. (d) Quantification of c. For each experimental condition, shown is EEA1 intensity (mean gray value) per cell calculated using Fiji relative to the highest intensity recorded. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: nEtOH = 68 cells; nE2 = 39 cells; FN: nEtOH = 94 cells; nE2 = 108 cells). (e) Confocal images of MCF7 cells seeded on FN treated for 15 min as indicated and stained for EEA1. Merges between differential interference contrast (DIC) microscopy and the green channel are shown. Arrows indicate early endosomes present either in the nuclear membrane or inside the nucleus. (f) Quantification of e. For each experimental condition, structures of 10–15 pixels in diameter were quantified using Fiji. Shown is the number of nuclear early endosomes per cell. It was calculated as the total number of EEA1+ vesicles in the nuclear membrane or inside the nucleus, per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: nEtOH = 59 cells; nE2 = 54 cells). (g) Top: Outline of the protocol followed and Western blot of a subcellular fractionation of MCF7 cells treated as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/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) Top: Western blot of a subcellular fractionation of MCF7 cells treated for 15 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test (n = 3 replicates). (i) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons (n = 3 replicates). Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Treatments: ethanol (vehicle) or 10−8 M E2, 2.5 µg/ml filipin, 5 µM PAO. Shown data are representative of at least three independent experiments. Black arrowheads indicate positions of 50-kD markers. White arrowheads indicate positions of 37-kD markers. Bars, 10 µm.$

E₂ stimulates endocytosis of vesicles containing ERα. (a) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for ERα. Arrows indicate ERα⁺ endosomes. (b) Quantification of a. For each experimental condition, structures of ∼200-nm diameter (10–15 pixels) were quantified using Fiji. Shown is the number of ERα⁺ puncta per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n_vehicle = 81 cells, n_E2 = 87 cells; FN: n_vehicle = 50 cells, n_E2 = 64 cells). (c) Confocal images of MCF7 cells seeded on BSA (left) or FN (right) treated for 15 min as indicated and stained for EEA1. Cells are delineated in white. Arrows indicate early endosomes. (d) Quantification of c. For each experimental condition, shown is EEA1 intensity (mean gray value) per cell calculated using Fiji relative to the highest intensity recorded. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: BSA: n_EtOH = 68 cells; n_E2 = 39 cells; FN: n_EtOH = 94 cells; n_E2 = 108 cells). (e) Confocal images of MCF7 cells seeded on FN treated for 15 min as indicated and stained for EEA1. Merges between differential interference contrast (DIC) microscopy and the green channel are shown. Arrows indicate early endosomes present either in the nuclear membrane or inside the nucleus. (f) Quantification of e. For each experimental condition, structures of 10–15 pixels in diameter were quantified using Fiji. Shown is the number of nuclear early endosomes per cell. It was calculated as the total number of EEA1⁺ vesicles in the nuclear membrane or inside the nucleus, per cell. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n_EtOH = 59 cells; n_E2 = 54 cells). (g) Top: Outline of the protocol followed and Western blot of a subcellular fractionation of MCF7 cells treated as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/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) Top: Western blot of a subcellular fractionation of MCF7 cells treated for 15 min as indicated. Blotting antibodies are shown on the left. Bottom: Densitometry. For each subcellular fraction, shown is the ERα/β-actin density ratio normalized to the control group. Differences between groups were analyzed by one-tailed paired Student’s t test (n = 3 replicates). (i) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons (n = 3 replicates). Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Treatments: ethanol (vehicle) or 10⁻⁸ M E₂, 2.5 µg/ml filipin, 5 µM PAO. Shown data are representative of at least three independent experiments. Black arrowheads indicate positions of 50-kD markers. White arrowheads indicate positions of 37-kD markers. Bars, 10 µm.