Fig. 5

O-GlcNAcylation of eIF2α suppresses HO-1 protein translation. A Immunofluorescence of HO-1 and DAPI was conducted in BT-549 cell spheres subjected to ArgS for 24 h (n = 3; left panel). The relative HO-1 intensity (right panel) was calculated by dividing the integrated HO-1 intensities by the selected sphere area. B The levels of HMOX1 mRNA were analyzed by qRT-PCR in BT-549 and MDA-MB-231 cells and the activity of the HMOX1 promoter was analyzed by bioluminescent reporter assay in MDA-MB-231 cells. The cells were harvested for total RNA extraction or the bioluminescent reporter assay and subjected to ArgS for 24 or 48 h, respectively (n = 3). qRT-PCR was performed using gene-specific primer pairs and analyzed by the 2^−∆∆Ct method. ΔCt refers to the difference in cycle threshold values between the target gene and reference gene. In the bioluminescent reporter assay, cells were transiently transfected with the HMOX1 ARE-Firefly luciferase (FLuc) reporter construct and the pRL-SV40 Renilla luciferase construct. The intensities of firefly luminescence were normalized with the Renilla luminescence intensities and presented as histograms. C Immunoblot analysis of HO-1 in BT-549 cells subjected to ArgS ± MG132 (10 μM) for 24 h (left panel). Endogenous and FLAG-tagged HO-1 were visualized using an anti-HO-1 antibody. Actin served as a loading control and ATF4 as an indicator of ER stress. The relative HO-1 protein level (right panel) was calculated by normalizing with the level in the control (+ Arg, -MG132), which was set as 1 after normalization with actin. D Immunoprecipitation coupled with Western blot analysis of de novo HO-1 synthesis upon ArgS in BT-549 cells overexpressing WT or O-GlcN-mut eIF2α (n = 3). The cells were cultured under ± Arg for 24 h and AHA (250 μM) was added for the last 6 h. The de novo synthesized HO-1 protein level was determined by comparing the densitometric tracing of HO-1 signal in the experimental conditions with the reference HO-1 signal (eIF2α WT; + Arg). The value of reference HO-1 was set as 1 after normalization with H3 (serving as a loading control). AHA-labeled proteins were detected using Click-iT chemistry, and the products were purified with streptavidin beads affinity pulldown. E Immunoprecipitation coupled with Western blot analysis of de novo ATF4 synthesis upon ArgS in BT-549 cells overexpressing WT or O-GlcN-mut eIF2α. Cells were exposed to Arg or not for 6 or 24 h with the addition of AHA (250 μM) for the last 6 h prior to harvesting. The pulled-down de novo AHA-labeled ATF4 proteins were then visualized through immunoblotting. F O-GlcN-mut relieved the ArgS-repressed de novo protein synthesis beyond HO-1 in BT-549 cells overexpressing either WT or O-GlcN-mut eIF2α. The cells were cultured under ± Arg for 24 h with the addition of AHA (250 μM) for the last 6 h, and protein input was visualized through Coomassie blue staining (left panel) and global de novo protein analysis was visualized using an anti-biotin antibody (right panel). A–E The data is presented as mean ± s.e.m. and was analyzed using either one-way ANOVA (for A–C) or two-way ANOVA followed by Tukey's multiple comparison test (for D, E). Statistical significance was determined with *p < 0.05; **p < 0.01; ***p < 0.001