Fig. 2: NSC80997 and NSC255112 induce Tn expression in human cell lines.

a Line graph shows metabolic activity of HEK^WT cells treated with 0–128 µM NSC80997 and NSC255112 for 24 h measured by MTT assay. Data are presented as average 595 nm absorbance values ± SEM from three independent experiments. b Representative images show HeLa^WT cells treated for 24 h with DMSO or 10 µM NSC80997 and stained with VVA lectin (green) and DAPI (magenta). Scale bar represents 20 µm. c Bar diagrams show cell surface Tn induction in HEK^WT cells treated with increasing concentrations of NSC80997 or DMSO control for 24 h. Binding of VVA (left) or anti-Tn (1E3) monoclonal antibody (right) was quantified by flow cytometry. Data from three independent experiments are presented as average mean fluorescence intensity (MFI) ± SEM (arbitrary unit) ***P < 0.0001 (ANOVA). d Bar diagrams show MFI values of cell surface VVA binding to MCF7, AGS, SH-SY5Y, and HeLa cells treated with 0-20 µM NSC80997 for 24 h assessed by flow cytometry. Data is representative of two independent experiments. e, f Time-course analysis of cell surface Tn induction by NSC80997 (e) and recovery of glycosylation after removal of NSC80997 from culture medium (f) in HEK293^WT cells (top) and HeLa^WT cells (bottom). Cells were treated with 10 µM NSC80997 and VVA binding was assessed at indicated timepoints by flow cytometry. Data from three independent experiments are presented as average mean fluorescence intensity (MFI) ± SEM (arbitrary units). ***P < 0.0001 (ANOVA) (e). Loss of surface Tn structures in HEK^WT (top) and HeLa^WT (bottom) cells after treatment with NSC80997 for 24 h and extensive washing was detected by VVA staining and flow cytometry analysis 24, 48, and 72 h after treatment (f). Data from three independent experiments are presented as average mean fluorescence intensity (MFI) ± SEM (arbitrary units). ***P < 0.0001 (ANOVA).