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. 2020 Feb 27;53:102693. doi: 10.1016/j.ebiom.2020.102693

Fig. 5.

Fig 5

O-GlcNAc promotes the activation of NF-kappa B in AIEC LF82-infected cells. (a) HCT116 cells were pretreated with 10 µM DON for 6 h before exposure to AIEC LF82 (MOI = 10) for additional 4 h. Whole cell extracts were analyzed with immunoblots for O-GlcNAc, IKKβ, and NF-κB (p65) with respective antibodies. GAPDH serves as a loading control. (b) IKKβ and NF-κB (p65) in HCT116 cells treated with DON and AIEC LF82 were immunoprecipiated with anti-IKKβ or anti-NF-κB (p65) antibody. The O-GlcNAcylated IKKβ and NF-κB (p65) were detected with an O-GlcNAc monoclonal antibody, RL2. (c) HCT116 cells were transfected with pcDNA3/His-p65 for 48 h. Cells were then treated with 10 µM DON for 6 h before exposure to AIEC LF82 (MOI = 10) for additional 4 h. The cells were fixed and stained with anti-p65 antibody for the immunofluorescence analysis. (d) HCT116 cells were pretreated with 10 µM DON for 6 h before exposure to AIEC LF82 (MOI = 10) for additional 4 h. Cytosolic and nuclear components of the cells were isolated for the western blot analysis. (e) HCT116 cells were transduced with pGL3/NF-κB and pRL (Renilla A luciferase vector) for 24 h. Cells were then treated with 10 µM DON for 6 h before exposure to AIEC LF82 (MOI = 10) for additional 4 h. After the treatment, cells were harvested for luciferase activity assay using Dual-Luciferase Reporter Assay System (Promega). Relative luciferase unit (RLU) was the ratio of NF-κB luciferase activity to Renilla A activity. ** P < .01 (Student's t-test). (f) HCT116 cells were pretreated with 10 µM DON for 6 h before exposure to AIEC LF82 (MOI = 10) for additional 4 h. After the treatment, supernatants of the cell culture were collected and centrifuged for the measurement of IL-1 and IL-6. * P < .05 and ** P < .01 (Student's t-test). (g, h) HCT116/GFP-LC3 cells were treated with DON and AIEC LF82 as described above. The cells were then analyzed with an Olympus IX51 fluorescence microscope for the GFP-LC3 punta representing autophagosome/autolysosome formation (g). The cell lysates were collected for the detection of autophagy markers LC3, p62, and ATG16. GAPDH was detected as a loading control (h).