Figure 3
ERα expression promotes an increase of autophagosomes and autophagic flux. (a) SK-01, SK-ERα, SK-ERβ and MCF-7 cells were transiently transfected with a GFP-RFP-LC3 fusion protein and treated with vehicle control and 500 nM of BafA1 for 6 h. In autophagosomes, both RFP and GFP signals are apparent whereas under acidic conditions when autophagosomes fuse with lysosomes just the RFP signal persists. In contrast, GFP signal diminishes due to a labile enhanced GFP under acidification. Representative images acquired by confocal microscopy are shown. Scale bars: 10 μm. (b) GFP-RFP-LC3-punctae were counted in all cell lines transfected with the ptfLC3 expression plasmid and treated with 500 nM of BafA1 for 6 h before fixation. At least six different positions of the corresponding stainings per experiment were documented utilizing confocal laser scanning microscopy and counted numbers of GFP-RFP-LC3 punctae were averaged. 59 SK-0 cells, 56 SK-ERα cells, 59 SK-ERβ cells and 58 MCF-7 cells were analyzed. Three independent experiments in each panel are expressed as mean±S.E.M. (*) on bars represents statistical significance of P<0.05 comparing two groups and n.s. displays no statistical significant difference. (c) Representative pictures of indirect immunofluorescence staining of endogenous LC3 (red) and SQSTM1 (green) in SK-01, SK-ERα, SK-ERβ and MCF-7 cells, treated with vehicle control and 500 nM of BafA1 for 6 h, are shown. DAPI (blue) was used to stain DNA. Scale bars: 10 μm. (d) Typical ultrastructure of SK-01, SK-ERα, SK-ERβ and MCF-7 cells visualized by using transmission electron microscopy. Magnifications of marked areas are shown in the panels to the right. ERα-expressing cells showed increased autophagic vesicles of different stages of maturation. Autophagosomes are indicated by A, autolysosomes by AL, nucleus by N and mitochondria by M
