Fig. 5: NFATc3 K384 deSUMOylation by SENP3 increases nuclear translocation of NFATc3 by decreasing its interaction with GSK-3β.

a–c Cytosolic and nuclear fractions were prepared and immunoblotting analyses with the indicated antibodies were performed. a. PANC-1 and AsPC-1 cells that stably expressed Flag-WT NFATc3 or Flag-NATc3 K384R were cultured under normoxia or hypoxia condition for 24 h. b PANC-1 and AsPC-1 cells with or without the expression of SENP3 sgRNA expressed Flag-WT NFATc3 or Flag-NFATc3 K384R proteins. The indicated cells were cultured under normoxia or hypoxia condition for 24 h. c Parental and the indicated SENP3-knockout AsPC-1 and PANC-1 cells were cultured under normoxia or hypoxia condition for 24 h. d AsPC-1 cells with or without the expression of SENP3 sgRNA expressed Flag-WT NFATc3 or Flag-NFATc3 K384R proteins. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. e ChIP assay with an anti-NFATc3 antibody and quantitative PCR with primers against the promoter regions of MYC in AsPC-1 cells with or without SENP3 depletion were performed. Two-sided t-test analyses were conducted. The data are presented as the means ± S.D. of three independent experiments (n = 3). **P < 0.01. f Flag-WT NFATc3 or Flag-NFATc3 K384R was expressed in AsPC-1 and PANC-1 cells with or without the expression of SENP3 sgRNA. The indicated cells were transfected with luciferase reporter plasmid (NFATc3-Luc) and stimulated with or without hypoxia for 12 h. The data are presented as the mean ± SD of triplicate samples (***P < 0.001; N.S. = not significant for the indicated comparison).