Fig 1: Presence of the calcineurin-NFAT pathway and the impact of cyclosporin A on FGF23 treatment.A) Transcript analysis of laser capture microdissected parathyroid glands from 3- week-old wild-type mice revealed presence of NFATC1-4 and the subunits for calcineurin A and B (CnA and CnB). Each bar represents one hybridization probe. Data is presented as mean (± SEM). B) Immunostaining for NFATC2 showed a partial nuclear translocation in PTH-KL-/- mice, and cytoplasmatic localization in wild-type mice after treatment with FGF23, indicating activation of the calcineurin-NFAT pathway in parathyroids of PTH-KL-/- mice. In addition, MCIP1, a facilitator of calcineurin signaling, was markedly higher in PTH-KL-/- compared to wild-type mice after treatment with FGF23 (10× and 40× magnification). C) Pretreatment of mice with the calcineurin inhibitor CsA followed by a single intravenous FGF23 injection. Wild-type mice had a preserved responsiveness to FGF23 as evidenced by a significant reduction in serum PTH 15 minutes after FGF23 injection (p<0.05) whereas FGF23-mediated inhibition of serum PTH was blunted in PTH-KL-/- mice. Figure depicts pooled data from two independent experiments, using 60 mg/kg and 150 mg/kg CsA respectively, with similar results. Total n = 10 for wild-type and 7 for PTH-KL-/- mice. *p<0.05. D) Relative change in PTH level in wild-type and PTH-KL-/- mice 15 min after a single FGF23 injection, with and without pretreatment of CsA. E) Thyro-parathyroid explants were cultured in serum-free medium and exposed to FGF23 for 2 h with or without pre-treatment by CsA. Wild-type and PTH-KL-/- mice responded with a similar decrease in PTH when exposed to FGF23. In contrast, in explants pre-treated with CsA (0.83 µM) for 2 h the response to FGF23 treatment was unaltered in wild-type mice but completely blunted in PTH-KL-/- mice. N = 5–10 for all groups. **p<0.01.