Fig. 2

The O-GlcNAcylation of eIF2α downregulates the antioxidant capacity, cell recovery and migratory ability of breast cancer cells in response to ArgS. A Immunoprecipitation coupled with immunoblot analysis was performed to measure the levels of GlcNAz-modified (O-GlcNAcylated) proteins in parental HEK 293T cells (n = 3; left panel). The O-GlcNAz-modified eIF2α level was calculated by normalizing the densitometric tracing of eIF2α signal to the H3 signal (right panel). The values were quantified using ImageLAB software and adjusted for background. B The same analysis was performed in WT and O-GlcN-mut, eIF2α overexpressing HEK 293 T cells (n = 2; left panel). The O-GlcNAz-modified eIF2α level was calculated as described in A, and the ratio in each experimental condition was compared to the reference (WT + Arg, set to 1; left panel). One representative immunoblot is shown for each analysis, with indicated antibodies. The black arrowheads indicate endogenous eIF2α, and the white arrowheads indicate FLAG-tagged eIF2α. C The levels of ROS were measured in BT-549 cells stably expressing WT or O-GlcN-mut eIF2α after 48 h of ArgS treatment (n = 3). The ROS levels were quantified using CellROX™ and flow cytometry. D The relative cell recovery was monitored in BT-549 cells stably overexpressing WT or O-GlcN-mut eIF2α over a 5-day period, after 24 h of ArgS treatment and replacement with complete medium. The cell recovery was determined using ACP assays and the data were normalized to the values of Day 0, set to 1. E The cell migration was measured in BT-549 cells stably overexpressing WT or O-GlcN-mut eIF2α after 24 h of ArgS treatment and replacement with complete medium. The cell migration was determined by calculating the % confluency within the gap area and was imaged and quantified using Image J software at 0, 12, 24, and 30 h. Data are presented as mean ± s.e.m. and analyzed using Two-Way ANOVA followed by Tukey's multiple comparison test. Statistical significance was determined with *p < 0.05; **p < 0.01; ***p < 0.001