Deacetylation of NDPK-D promoted its nuclear localization.

<p>(A) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. WT and 3KQ NDPK-D localized in both cytoplasm and nucleus, whereas the majority of 3KR mutants were retained in the nucleus. NDPK-D localization was classified in the transfected N1E cells. “C > N” indicates the cells predominantly exhibiting Myc-NDPK-D in the cytosol. “C = N” indicates the cells exhibiting cytosol and nuclear Myc-NDPK-D. “C < N” indicates the cells showing nuclear-accumulated Myc-NDPK-D. The percentage of transfected cells exhibiting indicated subcellular localization for NDPK-D is shown in the graph. Scale bar: 50 μm. **<i>P</i> < 0.01. n = 5. (B) N1E-115 cells were transfected with Myc-NDPK-D and fractionated into cytosol and nuclear fractions. Each fraction was subjected to deacetylation assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139616#pone.0139616.g004" target="_blank">Fig 4</a>. n = 4. (C) The HDAC inhibitor (TSA) seems to increase the acetylation level of NDPK-D in the cytosol fraction. n = 3. (D) Leptomycin B (LMB) treatment did not cause nuclear accumulation of NDPK-D. The cells transfected with Myc-tagged NDPK-D or HA-tagged zyxin (used as positive control) were cultured for 36 h, in the presence or absence of LMB during the last 6 h. The cells were stained with anti-Myc antibody for Myc-NDPK-D or anti-HA antibody for HA-zyxin. Scale bar: 50 μm. (E) SIRT1 causes nuclear accumulation of NDPK-D. N1E-115 cells were transfected with Myc-tagged WT NDPK-D and HA-tagged WT SIRT1 or deacetylase-deficient mutant SIRT1 (H363Y). After 36 h, cells were fixed and immunostained with anti-Myc and HA antibodies. NDPK-D localization was classified in N1E cells expressing both Myc-tagged NDPK-D and HA-tagged SIRT1. Scale bar: 20 μm. *<i>P</i> < 0.05, **<i>P</i> < 0.01. n = 4. (F) Acetylation-mimetic form of NDPK-D induced apoptosis. N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. Cells were immunostained with anti-cleaved caspase-3 and anti-Myc antibodies. The frequencies of cleaved caspase-3-positive cells in the transfected cells were normalized to that of control. **<i>P</i> < 0.01. n = 5. (G) The treatment with SIRT1 inhibitor EX527 tended to increase the number of cleaved caspase-3-positive cells. N1E-115 cells were treated with or without EX527, and cultured for 48 h. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s multiple comparison tests (A, D, E), or Welch’s t-test (B).