Fig 1. HEK293-ANKH^wt cells release ATP, which is rapidly converted into pyrophosphate (PPi) and AMP.

Detection of ANKH in HEK293 parental, HEK293-ANKH^wt and HEK293-ANKH^L244S cells by immunoblot analysis (A). Concentrations of pyrophosphate (PPi) (B) and AMP (C) were quantified enzymatically and followed in medium samples of HEK293 parental, HEK293-ANKH^wt and HEK293-ANKH^L244S cells over the course of 24 hours. ATP release by HEK293 parental, HEK293-ANKH^wt and HEK293-ANKH^L244S cells was followed in real time using a luciferase-based assay (D). Results of representative experiments performed in triplicate are shown. In panels B and C data are expressed as mean +/- SD. Panel D shows mean +/- SEM. Statistical significance for ANKH^wt C3 vs parental and ANKH^wt C3 vs ANKH^L244S C2, was calculated by 2-way ANOVA, with Bonferroni’s correction, using GraphPad Prism version 8.4.2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.