Fig. 6.

Calcineurin inhibits mTORC1 signaling to enhance NFATc1 activation. a mTORC1 signaling in osteoclast differentiation cultures 60 h after RANKL treatment with or without CsA, measured by S6K1 phosphorylation. p/T ratio of p-S6K1/total S6K1. b Representative images of TRAP-stained osteoclast differentiation cultures on day 9. c Western blotting of calcineurin from mTOR immunoprecipitates. d Bioinformatic analysis of PxIxIT and LxVP motifs in mTOR kinase. e, f NFATc1 mobility shifts in bone marrow osteoclast differentiation cultures 60 h after RANKL treatment. e Raptor^fl/fl-Lyz cKO mice. f Tsc1^fl/fl-Lyz cKO mice. g Western blot of NFATc1 from mTOR immunoprecipitates. h NFATc1 mobility shifts in bone marrow osteoclast differentiation cultures 48 h after RANKL treatment with or without rapamycin (Rapa) or CsA. i, j Expression of TRAP (i) and NFATc1 (j) during a time course of bone marrow osteoclast differentiation with rapamycin (Rapa) or CsA (n = 9). k A working model of how the calcineurin ⊣ mTORC1 ⊣ NFATc1 cascade regulates osteoclast differentiation. Error bars, SEM; *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; n.s. non-significant. Full-size scans of immunoblots are shown in Supplementary Fig. 10