KPNA1, KPNA5, and KPNA6 are required for E2-dependent PHB2 nuclear import in breast cancer cells.

<p>(A) Immunoblotting analysis was performed to evaluate the subcellular localization of endogenous PHB2 in the BIG3- and KPNAs (KPNA1, KPNA5, and KPNA6)-depleted MCF-7 cells. MCF-7 cells were treated with siBIG3 and each siKPNA, followed by E2 ± ERAP for 24 h. Then, the cells were separated into cytoplasmic and nuclear fractions; (B) The nuclear translocation of PHB2 in KPNA-depleted MCF-7 cells in the presence of E2 and ERAP was evaluated. MCF-7 cells were treated with each siKPNA followed by E2 ± ERAP for 24 h. Then, the cells were separated into cytoplasmic (Cyto) and nuclear (N) fractions. The data are expressed the fold increase over cytoplasm fraction of untreated siEGFP, siKPNA1, siKPNA2, siKPNA5 or siKPNA6-transfected cells (set at 1.0), respectively. (C) The relationship among each KPNA was evaluated in KPNA-depleted MCF-7 cells in presence of E2 and ERAP. α/β-Tubulin (tubulin) and lamin B1 (lamin) were used as loading controls for the cytoplasmic (Cyto) and nuclear (N) fractions, respectively (A, B). Full-length images of immunoblots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127707#pone.0127707.s003" target="_blank">S3A–S3C Fig</a>.</p

KPNA mediates the nuclear translocation of PHB2.

<p>(A) Immunoblotting analysis was performed to detect the subcellular localization of KPNA, ERα and PHB2. COS-7 cells co-transfected with HA-PHB2, each FLAG-KPNA and FLAG-ERα were treated with 10 nM E2 for 24 h and separated into cytoplasmic and nuclear fractions. Each KPNAs and ERα were detected by endogenous antibody. α/β-Tubulin (tubulin) and lamin B1 (lamin) were used as loading controls for the cytoplasmic (Cyto) and nuclear (N) fractions, respectively. (B) Representative immunofluorescence images of the subcellular localization of HA-PHB2 in COS-7 cells are shown; HA-PHB2 (red), DAPI (blue). (C) Statistical analyses of the nuclear intensity of translocated PHB2. The data represent the mean ± SD of four different points (***<i>P</i><0.001 in a two-sided Student’s <i>t</i>-test).</p

KPNA1, KPNA5, and KPNA6 induce E2-dependent nuclear translocation of PHB2.

<p>(A) Immunoblotting analysis was performed to evaluate the interactions between ERα and PHB2 in BIG3- and KPNA (KPNA1, KPNA5, and KPNA6)-depleted MCF-7 cells. MCF-7 cells were treated with siBIG3 and each siKPNA, followed by E2 ± ERAP for 24 h. Then, the nuclear fractions were immunoprecipitated with anti-ERα antibody and were immunoblotted with antibodies against the indicated proteins. The data are expressed the fold increase over E2-treated siBIG3-transfected cells of right and left panels, respectively (set at 1.0). ND: not detected. This experiment was performed using the nuclear fractions used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127707#pone.0127707.g003" target="_blank">Fig 3A</a>; (B) The interaction between ERα and PHB2 released by E2 and ERAP in the nuclear fractions was evaluated. MCF-7 cells depleted of each KPNA were treated with E2 ± ERAP for 24 h, and the nuclear fractions were immunoprecipitated with anti-ERα antibody. The data are expressed the fold increase over E2-treated siEGFP-transfected cells of right and left panels, respectively (set at 1.0). This experiment was performed using the nuclear fractions used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127707#pone.0127707.g003" target="_blank">Fig 3B</a>; (C) The <i>TFF1</i> expression levels following treatment with siBIG3 and siKPNA were evaluated using real-time PCR. The data are expressed as the fold increase over the untreated cells (set at 1.0) and represent the means ± SD of two independent experiments (**<i>P</i><0.01, ***<i>P</i><0.001 in a two-sided Student’s <i>t</i>-test); (D) Immunoblotting analysis was performed to identify the KPNA-binding regions in PHB2. The lysates from COS-7 cells transfected with the indicated HA-PHB2 constructs and FLAG-KPNAs were immunoprecipitated with an anti-FLAG antibody; (E) Immunoblotting analysis was performed to identify the BIG3-binding region in PHB2. The lysates from HEK293T cells transfected with the indicated HA-PHB2 constructs and FLAG-BIG3 were immunoprecipitated with an anti-FLAG antibody. Full-length images of immunoblots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127707#pone.0127707.s006" target="_blank">S6A–S6D Fig</a>.</p

The mechanism of the E2-dependent nuclear translocation of PHB2 through KPNAs in breast cancer cells.

<p>The mechanism of the E2-dependent nuclear translocation of PHB2 through KPNAs in breast cancer cells.</p

PHB2 interact with KPNAs.

<p>(A) The expression levels of the KPNA family of proteins in breast cancer cell lines and normal human mammary gland tissue were evaluated using semi-quantitative RT-PCR. <i>ACTB</i> is used as an internal control; (B, C) Immunoblotting analysis was performed to assess the interactions between PHB2 (B) or PHB2 NLS mutants (C) and each KPNA. The lysates from COS-7 cells transfected with PHB2 or PHB2 NLS mutants and each KPNA were immunoprecipitated with anti-FLAG antibody. Full-length images of immunoblots and semi-quantitative RT-PCR are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127707#pone.0127707.s001" target="_blank">S1A–S1C Fig</a> The data are expressed the fold increase over HA-PHB2 which bound to KPNA1 (set at 1.0) (B).</p

Antibody profiles for MuSK, LRP4, and clustered AChR in patients with MG seronegative for AChR antibody on radioimmunoprecipitation assay.

<p>LEMS, Lambert-Eaton myasthenic syndrome; MND, motor neuron disease; MG, myasthenia gravis; MuSK, muscle-specific tyrosine kinase; CBA, cell-based assay; RIPA, radioimmunoprecipitation assay; LRP4, low-density lipoprotein receptor-related protein 4; cAChR, clustered acetylcholine receptor.</p

Clinical features of the patients with the LRP4 antibody, and of the patients who were double seropositive.

<p>Clinical features of the patients with the LRP4 antibody, and of the patients who were double seropositive.</p

Clinical features of AChR antibody seronegative generalized MG (on radioimmunoprecipitation assay) according to the antibody profile.

<p>Clinical features of AChR antibody seronegative generalized MG (on radioimmunoprecipitation assay) according to the antibody profile.</p

Cell based assays to detect antibodies to clustered AChR, MuSK or LRP4.

<p><b>Patient IgG binding is shown in red. For the AChR and MuSK assays, EGFP tags exhibit a green fluorescence highlighting the transfected cells; there is no color tag in the LRP4 assay. Patient 41 is positive for AChR, Patient 42 is positive for MuSK antibodies, and patient 72 is low positive for LRP4 antibodies.</b> MuSK, muscle-specific tyrosine kinase; LRP4, low-density lipoprotein receptor-related protein 4; AChR, acetylcholine receptor.</p

Statistical values of Rio (RS) and modified Rio (MRS) scores.

<p>Statistical values of Rio (RS) and modified Rio (MRS) scores.</p