Fig. 5
Cell surface localization of CLEC10A ligands and analysis of the expression of different components of the O-glycosylation machinery (a) Cell surface proteins of MCF7 and T47D cells were biotinylated with non-cell-permeable sulfo-NHS-SS-biotin after 48 h treatment by Tam (4 μM), Zeocin (Zeo; 250 μg/ml) and hydrogen peroxide (H2O2; 30 μM), respectively. Non-treated cells (n) and cells treated by ethanol (EtOH) served as controls. After cell lysis, biotinylated surface proteins were precipitated by streptavidin agarose. Western blot analyses were performed with CLEC10A and monoclonal antibodies directed against Her2/neu (ERBB2), and E-cadherin; C.S.: cell surface. b ROS measurements in MCF7 and T47D cells after 2.5 h and 48 h, respectively. FITC intensities of the cells were measured in quadruplicates by flow cytometry. Signals of unstained cells served as background controls and were subtracted from signals from stained cells. Results of stained, treated cells (E, T, Z, H) were normalized to stained, non-treated cells (N). The averages and standard deviations were calculated and Student’s t-test was performed for determination of significance. *** P < 0.001, **** P < 0.0001. c Western blot analysis of proteins LC3b and p62 involved in autophagy in Tam, zeocin and hydrogen peroxide treated cells. Untreated (n) and ethanol (EtOH) treated cells served as controls. MCF7 and T47D cells were treated for 48 h, lysed, and 20 μg of total protein was subjected to SDS-PAGE. β-actin served as loading control. d Western Blot analysis of γH2A.X as a marker for DNA damages. β-actin served as loading control. e Western blot analysis of levels of MUC1, T-synthase, COSMC and Beclin 1 protein expression in Tam, zeocin and hydrogen peroxide treated MCF7 and T47D cells in comparison to untreated controls (n). Cells were treated as described above. β-actin served as loading control