Fig. 3.

2-DG reduces IFN-γ-induced JAK-STAT1 pathway activation by reducing ATP production.

(a) Immunoblot analysis of specified proteins in BMDMs as a function of time of IFN-γ stimulation ±1 hour pre-treatment with 2-DG (10 mM). Data are representative of two independent experiments.

(b) Immunofluorescence staining of p-STAT-1 in BMDMs after incubation with IFN-γ for 30 min ± 1 hour pre-treatment with 2-DG (10 mM). Data are representative of two independent experiments.

(c) to (e) ATP production, ADP/ATP, AMP/ATP ratio of Raw264.7 cells, measured by HPLC after stimulation with IFN-γ for 30 min ± 1 hour pre-treatment with 2-DG (10 mM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS: no significant difference. Data are represented as mean ± sem (n = 6).

(f) Immunoblot analysis of specified proteins in Raw264.7 cells after stimulation with IFN-γ for 30 min ± 1 hour pre-treatment with indicated concentrations of 2-DG. Data are representative of two independent experiments,

(g) Immunoblot analysis of specified proteins in Raw264.7 cells after permeabilization with SLO (100 ng/ml), incubation with ATP (0–10 mM) for 15 min and 1-hour stimulation with IFN-γ (±10 mM 2-DG). Data are representative of two independent experiments.

(h) Immunoblot analysis of specified proteins in Raw264.7 cells after stimulation with IFN-γ for 9 h in medium containing different concentrations of glucose ±1 hour pre-treatment with JAK inhibitor I (100 nM). Data are representative of three independent experiments.

(i) Immunoblot analysis of specified proteins in Raw264.7 cells after stimulation with IFN-γ for 1 h in medium containing glucose or galactose ± oligomycin (1 μM). Data are representative of three independent experiments.