Fig. 6

ENT3 lysosomal adenosine transport regulates AMPK and autophagy signaling. Schematic representation of the activation of AMPK by extracellular and lysosomal pools of adenosine; Ado, adenosine; AMP, adenosine monophosphate (a). Mass spectrometry analysis of adenosine levels in lysosomes derived from mouse spleen lysates and HEK293 cells expressing GIPZ-shRNA (control) or ENT3 shRNA (n = 3, mean ± SEM) (b). HEK293 cells transduced with increasing MOI of hENT3 retroviruses were analyzed for lysosomal adenosine and cytosolic adenosine and AMP, and lysosomal acid phosphatase activity (n = 3, mean ± SEM) (c; above). Whole-cell lysates prepared from cells infected with the corresponding MOIs of hENT3 viruses were probed with the indicated antibodies. β-Actin served as the loading control (c; below). Quantification results from densitometric analysis of the immunoblots (n = 3, mean ± SEM) (d). Uptake of AICAR (20 μm) into Xenopus oocytes at 25 °C after 22 h of injection of N-terminal deleted ENT3 transcripts (ΔN36ENT3; to remove the lysosomal-targeting signal (shown in (h)) and enabling cell surface localization) was measured in transport buffer with pH 5.5. AICAR concentrations measured by mass spectrometry (n = 8 oocytes, mean ± SEM) (e). Mass spectrometry analysis of AICAR levels in lysosomes derived from MSCs derived from 12-week-old mice after the treatment of MSCs with AICAR (20 μm) for 4 h (n = 3, mean ± SEM) (f). HEK293 cells cultured in GFM were transfected with pE-YFP or pE-ENT3-YFP and treated with iodotubericidin (50 nM) for 4 h and lysates examined for indicated proteins. Iodotubericidin abrogates glucose starvation and ENT3-induced pAMPK activation. GFM, glucose-free medium (g). Schematic representation of WT ENT3 (ENT3) and localization-deficient ENT3 (ΔN36ENT3) mutants fused with YFP. A lysosomal-targeting motif in the N-terminus of ENT3 (red) and YFP tag (yellow) at the N-terminus are shown (h). HEK293 cells transduced with WT ENT3 but not N-terminal deleted ENT3 elicit glucose starvation-induced pAMPK activation (i). Statistical analyses were performed using two-tailed Student’s t-test. *P < 0.05. Source data are provided as a Source Data file