Fig. 4: STAT3 signaling induced by stimulation of β-AR increases the ATP/AMP ratio, resulting in downregulation of AMPK signaling in MDSCs.
In vitro generated WT + PBS and WT + ISO MDSCs as in Fig. 2 were treated with treated with 13C5-Gln and isotope-labeling levels in (A) ATP and (B) AMP were compared at 2 h, 8 h and 20 h. post treatment by Ion Chromatography-Ultra High-Resolution Mass Spectrometry (IC-UHRMS) as unlabeled (0), isotope labeled (C*), and total (pool of 0 + C*) (***p = 0.0001; ***p = 0.0002). Two-way ANOVA with Tukey’s multiple comparison tests. Data are presented as mean ± SEM. Representative of two independent experiments with n = 3 technical replicates per group. C The ATP/AMP ratio in WT + ISO compared WT + PBS control MDSCs was calculated based on the total ATP and AMP levels in the tracing experiment. (*p = 0.0120; *p = 0.0182) Data are presented as mean ± SEM. Representative of two independent experiments with n = 3 technical replicates per group. D Activation levels of signaling markers related to the AMPK pathway were determined post ISO treatment treated (WT + ISO) and control (WT + PBS) MDSCs using phospho-antibody array. Phosphorylation levels were measured for pAMPKbeta1 (Ser182), pMAP3K7/TAK1 (Thr184), p4E-BP1 (Thr45), pACC1 (Ser80), pP70S6K (Thr421), pP70S6K (Ser424), pPDK1 (Ser241), pCyclin D1 (Thr286), pEEF2 (Thr56), pelF4E (Ser209), pPP2A-alpha (Tyr307), pFOXO1/3/4-pan (Thr24/32). (*p = 0.0124) The paired t tests. Data are presented as mean ± SEM. n = 12 target protein per group. E, F Western blot analysis demonstrating AMPK and pAMPK (Tyr172) levels in generated MDSCs, treated with the STAT3 signaling blocker, JSI-124 (1 µM). Derived from the same samples in Fig. 3E, F. Results are representative of three independent experiments with n = 1 per group. Source data containing independent experiments raw data are provided as a Source Data file.
