Fig. 2: Glycogenolysis-derived G6P is channeled to the PPP.

a, b Pygl and Pygm expression in untreated, IFN-γ/LPS or IL-4 treated BMDMs were determined by real-time PCR (a) and western blot (b). c BMDMs differentiated in normal ¹²C-glucose were stimulated with IFN-γ/LPS or IL-4 for 6 h and switched to ¹³C-glucose for 6 h, LC-MS/MS was performed for m + 5-labeled R5P. d BMDMs were pretreated with GPI for 30 min or pretransfected with siRNA (Gys1 or Pygl) for 24 h prior to stimulation with IFN-γ/LPS for 6 h and switched to ¹³C-glucose for 6 h, LC-MS/MS was performed for m + 5-labeled R5P. e, f G6pdx and 6Pgd expression in untreated, IFN-γ/LPS or IL-4 treated BMDMs were determined by real-time PCR (e) and western blot (f). g Pygl or G6pdx siRNA transfected BMDMs were stimulated with IFN-γ/LPS for 36 h. Intracellular glycogen levels were detected by colorimetric assay. h BMDMs differentiated in normal ¹²C-glucose were stimulated with IFN-γ/LPS or IL-4 for 6 h and switched to ¹³C-glucose for 6 h, LC-MS/MS was performed for m + 7-labeled S7P, m + 5-labeled S7P and m + 4-labeled E4P. i, j BMDMs cultured and differentiated in ¹³C-glucose were pretreated with GPI for 30 min prior to stimulation with IFN-γ/LPS for 6 h and switched to ¹²C-glucose for 2 or 4 h, ¹³C-labeled G6P/G1P were detected by LC-MS/MS (i). The ratio of G6P/G1P from glycogenolysis or glycolysis was calculated. The glycogenolysis-derived G6P using the format of [m + 6 G6P (IFN-γ/LPS)–m + 6 G6P (IFN-γ/LPS + GPI)]/Total G6P (IFN-γ/LPS) and the glycolysis-derived G6P by the format of [m + 0 G6P (IFN-γ/LPS)] / Total G6P (IFN-γ/LPS) (j). Unless otherwise specified, n = 3 biologically independent experiments were performed. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA.