Figure 4
E2 or ICI treatment does not alter autophagic flux or expression of key autophagy pathway-related proteins. (a) SK-01, SK-ERα, SK-ERβ and MCF-7 cells were incubated with 10 nM E2 and 1 μM ICI or vehicle control for 24 h. Six hours before cell lysis indicated cells were additionally treated with 500 nM BafA1 and subjected to western blot analysis. Autophagic flux was determined by the accumulation of LC3-II. Therefore, normalized LC3-II levels in the absence of the lysosomal inhibitor were subtracted from the corresponding levels obtained in the presence of BafA1. Tubulin was used as a loading control. E2 or ICI treatment does not affect autophagic flux in each of the analyzed cell types. (b) Cells were treated with E2 or ICI for 24 h and western blot analysis of indicated proteins was performed. Tubulin served as a loading control. Except of the pmTOR/mTOR ratio of E2 and ICI treatment of SK-ERβ there was no effect on protein expression of pmTOR/mTOR, WIPI1, PI3KCIII, ATG7 and BAG3 after drug treatment. (a, b) Values of threre independent experiments in each panel are expressed as mean±S.E.M. and control SK-01 cells were set to 100%. (§) on bars represents statistical significance of P<0.05 comparing SK-ERα or SK-ERβ cells with SK-01 cells with the same treatment. (#) on bars represents statistical significance of P<0.05 comparing E2- or ICI-treated SK-ERα cells with E2- or ICI-treated SK-ERβ cells. (‡) on bars displays a statistical significance of P<0.05 of E2-treated SK-ERβ cells compared with ICI-treated SK-ERβ cells. All other combinations show no statistical significant difference
