The involvement of lysosomes in E2-induced ERα activities.

<p>(A) RT-qPCR analysis of pS2/TIFF (pS2), cathepsin D (CatD) and progesterone receptor (PR) mRNA expression normalized on the GAPDH mRNA expression in MCF-7 cells treated with E2 (10 nM) for 24 hrs both in the presence and in the absence of chloroquine (Clo–10 µM) treatment. Western blot (B) and relative densitometric (B’) ERK1/2 and AKT phosphorylation in MCF-7 cells treated with E2 (10 nM) at different time points. Where indicated, cells were treated chloroquine (Clo–10 µM) 30 min before E2 administration. Loading control was done by evaluating vinculin expression in the same filter. *indicates significant differences with respect to the relative control (0) sample; °indicates significant differences with respect to the corresponding E2 sample (<i>p</i><0.05). Figure shows representative blots of three independent experiments.</p

The involvement of lysosomes in E2-induced ERα degradation in MCF-7 cells.

<p>(A) XTT assay in growing MCF-7 cells treated for 24 hrs with different doses of chloroquine (Clo). (B) Western blot and relative densitometric analyses (C’) of ERα cellular levels in MCF-7 cells treated for 2 hrs with E2 (10 nM) in the presence of different concentrations of chloroquine (Clo). (C) Western blot and relative densitometric analyses (C’) of EGF-R cellular levels in HeLa cells treated for 2 hrs with EGF (1 µg/ml) in the presence of different concentrations of chloroquine (Clo). (D) Western blot and relative densitometric analyses of ERα cellular levels in MCF-7 cells treated with chloroquine (Clo 10 µM) in the presence of different doses of E2. (E–F) Western blot and relative densitometric analyses of ERα cellular levels in MCF-7 cells treated for with E2 (10 nM) at different time points in the presence of chloroquine (Clo 10 µM). Inhibitor alone was administrated for 2 hours and 30 min. Loading control was done by evaluating vinculin expression in the same filter. *indicates significant differences with respect to the control sample (0 or -); °indicates significant differences with respect to the corresponding E2-treated or EGF-treated samples (<i>p</i><0.05). Figure shows representative blots of at least three independent experiments.</p

ERα localization to lysosomes.

<p>(A) Growing MCF-7 cells were co-stained with anti-ERα Sp-1 and D-12 antibodies. (B) Growing pc DNA flag ERα -transfected HeLa cells were co-stained with anti-ERα HC-20 and flag antibodies. (C) Western blot analysis of ERα cellular levels in growing MCF-7 cells treated with cycloheximide (CHX - 1 mg/ml) for 24 hrs with both anti-ERα Sp-1 and D-12 antibodies. Loading control was done by evaluating vinculin expression in the same filter. (D) Anti-ERα Sp-1 staining of MCF-7 cells treated for with cycloheximide (CHX - 1 mg/ml) for 24 hrs. MCF-7 cells were co-stained with anti-ERα Sp-1 antibody together with either LAMP-2 antibody (E) or lysotracker (F) both in the presence and in the absence of E2 (10 nM–2 hrs). pc DNA flag ERα (Nessi)-transfected HeLa cells were co-stained with anti-ERα HC-20 antibody together with either LAMP-2 antibody (G) or lysotracker (H) both in the presence and in the absence of E2 (10 nM–2 hrs). Figures show one unique confocal plane. All co-staining procedures were described in details in the Material and Methods section.</p

The involvement of 26S proteasome in E2-induced ERα degradation in MCF-7 cells.

<p>(A) Western blot of ERα, p53, and ubiquitin (Ub) cellular levels in MCF-7 cells treated for 2 hrs with E2 (10 nM) in the presence of different concentrations of the 26S proteasome inhibitor (Mg-132). (B) Western blot of EGF-R, and ubiquitin (Ub) cellular levels in HeLa cells treated for 2 hrs with EGF (1 µg/ml) in the presence of different concentrations of the 26S proteasome inhibitor (Mg-132). (B’) Densitometric analyses related to ERα and EGF-R protein levels in panel A and B, respectively. (C) Western blot of ERα (high and low exposures) and ubiquitin (Ub) cellular levels in MCF-7 cells treated with the 26S proteasome inhibitor (Mg-132 1 µM) in the presence of different doses of E2. Densitometric analyses of ERα high exposure were performed. (D) Western blot of ERα, p53, and ubiquitin (Ub) cellular levels in MCF-7 cells treated for with E2 (10 nM) at different time points in the presence of the 26S proteasome inhibitor (Mg-132 1 µM). Inhibitor alone was administrated for 2 hours and 30 min. Loading control was done by evaluating vinculin expression in the same filter. *indicates significant differences with respect to the control sample (0 or -); °indicates significant differences with respect to the corresponding E2-treated samples (<i>p</i><0.05). Figure shows representative blots of at least three independent experiments.</p

The involvement of lysosomes in E2-induced cell proliferation.

<p>Western blot (A) and relative densitometric (A’) analyses of cyclin D1 (Cyc D1) and Bcl-2 expression levels in MCF-7 cells treated with E2 (10 nM–24 hours) both in the presence and in the absence of chloroquine (Clo–10 µM). Loading control was done by evaluating tubulin expression in the same filter. *indicates significant differences with respect to the relative control (−) sample; ° indicates significant differences with respect to the corresponding E2 sample (<i>p</i><0.05). Figure shows representative blots of three independent experiments. (B) RT qPCR analysis of cyclin D1 (Cyc D1) mRNA expression normalized on the GAPDH mRNA expression in MCF-7 cells treated with E2 (10 nM–24 hours) both in the presence and in the absence of chloroquine (Clo–10 µM). (C) Number of MCF-7 cells treated with E2 (10 nM–48 hours) both in the presence and in the absence of chloroquine (Clo–10 µM). *indicates significant differences with respect to control (−); °indicates significant differences with respect to E2 sample (<i>p</i><0.01).</p