FIGURE 4.

2‐Deoxy‐D‐glucose (2‐DG) inhibits the functions of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and interferon (IFN)‐γ. (A) HeLa cells were transfected with Stat3 Luc or NF‐κB Luc reporter plasmids or phRL‐TK (internal control). After 24 h, cells were treated with or without 25 mM 2‐DG for 8 h and then with 0.4 µg/mL IL‐6 with soluble IL‐6 receptor (sIL‐6R) (Stat3 Luc), 50 ng/mL IL‐1β (NF‐κB Luc), or 100 ng/mL TNF‐α (NF‐κB Luc) for 4 h. After stimulation, relative luciferase activity was evaluated. Results were analysed using one‐way ANOVA followed by Tukey's post hoc test. ****p < .0001, ***p < .001, **p < .01 and *p < .05. n.s.; not significant. Graphs are presented as the mean ±s.d. (n = 3). (B) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. The band shift of TNF receptor 1 (TNFR1) was determined by immunoblotting. Glycopeptidase F (GP‐F) extract was used as a control for inhibition of N‐linked glycosylation. (C) Binding of TNF‐α to its receptor in the presence or absence of 2‐DG was determined using FITC‐conjugated TNF‐α by flow cytometric analysis of THP1 cells (left panel). The same cells were subjected to immunoblotting using an anti‐TNFR1 antibody as described in (B). (D) Wildtype mouse embryonic fibroblasts (MEFs) were stimulated with 3000 U/mL IFN‐γ for the indicated times. Activating phosphorylation of Janus kinases (JAK1, JAK2, and TYK2) was assessed by immunoblotting. (E) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. Band shift of IFN‐γ receptor α‐chain (IFNGRα) was assessed by immunoblotting. Tunicamycin and GP‐F extracts were used as controls for inhibition of N‐linked glycosylation