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. 2017 Jan 3;114(3):504–509. doi: 10.1073/pnas.1615072114

Fig. 2.

Fig. 2.

TRAIL induces calcineurin-mediated dephosphorylation of Dyn1 and TRAIL–DR endocytosis to reduce apoptosis. (A) TRAIL binding to WT and Dyn1KO A549 cells measured at 4 °C. (B) Effects of calcineurin inhibition by CsA (20 µM) on TRAIL activity in A549 WT and Dyn1KO cells. (C) TRAIL activity (100 ng/mL) in A549 WT, Dyn1KO, and Dyn1KO reconstituted with Dyn1 WT (KO + WT) or the nonphosphorylatable mutant, Dyn1 S774A/S778A (KO + AA), in the presence or absence of CsA. (D) FLAG-tag TRAIL uptake in control and CsA pretreated cells. (E) TfnR uptake in the presence or absence of CsA. (F) Caspase-3/7 activation in TRAIL-treated MDA-MB-231 cells in the presence or absence of CsA. (G) Dyn1 phosphorylation (pSer774) profiles upon TRAIL stimulation (100 ng/mL) in MDA-MB-231 cells in the presence or absence of CsA, and quantification of pS774-Dyn1/total-Dyn1. (H) TRAIL-induced apoptosis in MDA-MB-231 WT and Dyn1 overexpressing cells in the presence or absence of CsA. All internalization rates were calculated relative to surface bound TfnR or FLAG-tag TRAIL. Reduction in cell viability by TRAIL (%) was calculated relative to control wells incubated with solvent alone or 20 µM of CsA. All of the results are mean values ± SD (n = 3). Two-tailed Student’s t tests were used to assess statistical significance. *P < 0.05, **P < 0.005, ***P < 0.0005; ns, nonsignificant.