Figure 2. ERα is degraded in lysosomes... | Rockefeller University Press

Figure 2.

$Figure 2. ERα is degraded in lysosomes and rescued by FN. (a) Top: Western blot of T47D cells seeded on BSA or FN pretreated with BZ 8nM or its vehicle (saline) for 4 h and treated as indicated. Bottom: Densitometry. For each subcellular fraction, the mean ERα/β-actin density ratio is shown normalized to the mean control group. (b) Top: Western blot of a subcellular fractionation of T47D cells pretreated for 90 min with 25 nM BAF or its vehicle (DMSO) and then treated for 60 min with 10−8 M E2 or its vehicle (ethanol). Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, the ERα/β-actin density ratio is shown, 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). (c) Confocal images of T47D cells expressing GFP-Rab7 seeded on BSA treated for 60 min with vehicle or E2, and stained for ERα. In the inset, arrows indicate points of colocalization. (d) Quantification of c. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: nvehicle = 11 fields, nE2 = 12 fields; Manders’: nvehicle = 8 fields, nE2 = 9 fields). (e) Confocal images of T47D cells expressing GFP-Rab7 seeded on FN treated for 60 min with vehicle or E2, and stained for ERα. (f) Quantification of e. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: nvehicle = 9 fields, nE2 = 7 fields). Treatments: ethanol (vehicle) or 10−8 M E2, 8 nM BZ. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 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 (unless otherwise indicated).$

ERα is degraded in lysosomes and rescued by FN. (a) Top: Western blot of T47D cells seeded on BSA or FN pretreated with BZ 8nM or its vehicle (saline) for 4 h and treated as indicated. Bottom: Densitometry. For each subcellular fraction, the mean ERα/β-actin density ratio is shown normalized to the mean control group. (b) Top: Western blot of a subcellular fractionation of T47D cells pretreated for 90 min with 25 nM BAF or its vehicle (DMSO) and then treated for 60 min with 10⁻⁸ M E₂ or its vehicle (ethanol). Blotting antibodies are shown on the left. Bottom: Densitometry. For each experimental condition, the ERα/β-actin density ratio is shown, 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). (c) Confocal images of T47D cells expressing GFP-Rab7 seeded on BSA treated for 60 min with vehicle or E₂, and stained for ERα. In the inset, arrows indicate points of colocalization. (d) Quantification of c. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: Pearson’s: n_vehicle = 11 fields, n_E2 = 12 fields; Manders’: n_vehicle = 8 fields, n_E2 = 9 fields). (e) Confocal images of T47D cells expressing GFP-Rab7 seeded on FN treated for 60 min with vehicle or E₂, and stained for ERα. (f) Quantification of e. For each experimental condition, Pearson’s correlation index and Manders’ coefficients (M1 and M2) were calculated within the areas of colocalization using Fiji. Data are represented as mean ± SD. Differences between groups were analyzed by one-tailed Student’s t test (per replicate: n_vehicle = 9 fields, n_E2 = 7 fields). Treatments: ethanol (vehicle) or 10⁻⁸ M E₂, 8 nM BZ. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 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 (unless otherwise indicated).