Journal: Cell Communication and Signaling : CCS

Article Title: Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells

doi: 10.1186/s12964-019-0420-9

Figure Lengend Snippet: CLEC10A staining of various normal human tissues arranged on a tissue microarray. Protein domain histochemistry was performed after complexing of recombinant, myc-tagged CLEC10A with a biotinylated anti-myc antibody conjugated to streptavidin-horseradish peroxidase. 3,3′-diamino-benzidine (DAB) was used as chromogenic substrate and tissues were counterstained with hematoxylin. Tissues stained positive for CLEC10A are marked with “+“. Scale Bar: 100 μm. Inserts with higher magnification of representative tissue areas are given for breast, cervical and endometrium tissues (scale bar: 10 μm)

Article Snippet: For coupling, beads were diluted in 700 μl PBS, 0.1% BSA and 3 μg biotinylated Tn antigen or biotinylated spacer control (Lectinity) were added and incubated at 4 °C for 16 h. Beads were washed two times with PBS, 0.1% BSA and resuspended in 1 ml PBS, 0.1% BSA.

Techniques: Staining, Microarray, Recombinant

Journal: Cell Communication and Signaling : CCS

Article Title: Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells

doi: 10.1186/s12964-019-0420-9

Figure Lengend Snippet: Increased phagocytosis of CLEC10A ligands by macrophages. a Surface expression of CLEC10A and CD16 on macrophages derived from human PBMC of healthy donors after differentiation by M-CSF. Subsequent blocking of Fc-receptors, macrophages were stained by using an APC labeled anti-CLEC10A antibody and a PerCP-Cy5 labeled anti CD16-antibody, respectively (red filled histograms). Fluorescence intensity was compared to cells stained with the corresponding isotype controls (non-filled histogram). b Increased uptake of fluorospheres carrying Tn-antigen (red, filled histogram) in macrophages compared to control beads carrying the spacer (grey, filled histogram). As a control for calcium-dependent internalization, the uptake of Tn- and control beads was investigated in the presence of EDTA (red dotted line and grey dotted line). The different peaks are due to the uptake of distinct numbers of particles per macrophage. c Macrophages from two independent donors were incubated with CFSE-labelled MCF7 cells treated with 4 μM Tamoxifen (Tam) for 48 h or with cells treated with solvent control (−). To analyze engulfment of dead cells by macrophages, aliquots of labelled cells were treated by freeze and thaw cycles (− 80 °C). The amount of CFSE and CLEC10A double positive cells were measured by flow cytometry. Dot plots of four representative measurements are given. Error bars depict the standard deviation of three technical replicates. p values: donor 1 – vs. Tam = 0.000014; donor 1 – (− 80 °C) vs. Tam (− 80 °C) = 0,0061; donor 2 – vs. Tam = 0.0000035; donor 2 – (− 80 °C) vs. Tam (− 80 °C) = 0.0018

Article Snippet: For coupling, beads were diluted in 700 μl PBS, 0.1% BSA and 3 μg biotinylated Tn antigen or biotinylated spacer control (Lectinity) were added and incubated at 4 °C for 16 h. Beads were washed two times with PBS, 0.1% BSA and resuspended in 1 ml PBS, 0.1% BSA.

Techniques: Expressing, Derivative Assay, Blocking Assay, Staining, Labeling, Fluorescence, Control, Incubation, Solvent, Flow Cytometry, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells

doi: 10.1186/s12964-019-0420-9

Figure Lengend Snippet: 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 (H 2 O 2 ; 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

Article Snippet: For coupling, beads were diluted in 700 μl PBS, 0.1% BSA and 3 μg biotinylated Tn antigen or biotinylated spacer control (Lectinity) were added and incubated at 4 °C for 16 h. Beads were washed two times with PBS, 0.1% BSA and resuspended in 1 ml PBS, 0.1% BSA.

Techniques: Expressing, Glycoproteomics, Lysis, Western Blot, Bioprocessing, Flow Cytometry, Staining, SDS Page, Control, Marker, Comparison