Fig 2: NMDAR signaling enhances CPEB3–IPO5 interaction through Ran-related changes. (A) Neurons stimulated with 3 min of NMDA and then incubated for the denoted time were used for IP with CPEB3 and IPO5 IgGs, followed by immunodetection with IPO5, CPEB3 and Ran antibodies. The intensities of immunodetected signals analysed by the ImageJ software are displayed as relative ratios at the bottoms of blots. The ratios of the co-precipitated target versus the immunoprecipitated protein (IPO5/CPEB3 or CPEB3/IPO5) are shown in red. (B) The cytoplasmic fraction isolated from neurons treated with ± NMDA was precipitated with the RanGTP antibody and immunoblotted with Ran antibody. (C and D) HeLa cells expressing myc-CPEB3, EGFP-IPO5 along with Ran-wt, T24N or Q69L mutant were pulled down with (C) flag or (D) myc antibodies. The precipitated substances were probed with GFP, myc and flag antibodies. (E) Neurons stimulated with a pulse of NMDA or NMDA for the indicated times were harvested for western blotting using RanBP1 and tubulin antibodies. (F) The nuclear and cytosol lysates fractionated from neurons treated with ± NMDA for 30 min were used for immunoblotting. (G) Schematic model of NMDA-induced nuclear import of CPEB3. NMDAR signaling increases RanBP1 expression, combined with RanGAP to stimulate Ran’s GTPase activity. Consequently, the IPO5-RanGDP complex can bind to CPEB3 and deliver CPEB3 to the nucleus. After nuclear import, a high RanGTP concentration and RCC1 in the nucleus triggers the release of CPEB3 from the ternary complex because RanGTP impairs the interaction between IPO5 and CPEB3. The nuclear export of CPEB3 is mediated by CRM1.

Fig 3: XPO1 inhibition modulates differential Akt phosphorylation in TAM-resistant cells and tumor xenografts. (A) GSEA analysis of RNA-Seq data depicting regulation of Akt signaling by the 4-OHT+SEL combination. ERa-XPO1 targeting changed XPO1 and pAkt protein expressions in both TAM-sensitive and TAM-resistant breast cancer cell lines. Western blot analysis (B) and immunofluorescence analysis (* p < 0.05) (C) of BT474 cells treated with 4-OHT (10-6 M) and SEL (10-7 M) for 24 h alone and in combination. Protein expression was quantified and is shown in bar graphs. A one-way analysis of variance (ANOVA) model was used for statistical significance of treatment effect and values were presented as mean ± SEM from three independent experiments (* p < 0.05, ** p < 0.01). scale bar = 200 pixels. (D) IHC staining of pAkt T308 and pAkt S473 in tumor samples obtained from BT474 xenografts received individual and combined TAM and SEL treatments (from Figure 1C). In the figure, red-brownish staining represents pAkt T308 and pAkt S473 proteins. This figure showed that individual 4-OHT and SEL treatments activated two different phosphorylation sites of Akt protein. The top panel indicated that SEL treatment activated pAkt T308 phosphorylation in BT474 tumor xenografts. The bottom panel showed that TAM treatment activated pAkt S473 phosphorylation in BT474 tumor xenografts. Quantification of positive staining was performed digitally by selecting four independent regions on the sample slides and the results were given in the corresponding graphs. Statistical significance was presented as mean ± SEM by using one-way analysis of variance (ANOVA) model (** p < 0.01, **** p < 0.0001). scale bar = 20 µm.

Fig 4: Association of XPO1 Overexpression and NF-?B with Ki67. The IHC expressions of both XPO1 (A) and NF-?B (B) show highly concordance of immunoreactivity within the tissue sections expressed Ki67 (C and D respectively) (original magnification- 400x).

Fig 5: Aberrant nuclear export of HP1? is induced by high-risk HPV E6 protein.a Immunohistochemical staining of HP1? protein in cervical tissues from normal specimens (upper) and HPV-positive cervical adenocarcinoma patients (lower). b Immunoblot analysis of cytoplasmic and nuclear extracts from C33A cells expressing Flag-HPV16 E6. c Immunoblot analysis of cytoplasmic and nuclear extracts from SiHa cells expressing siRNA of HPV16 E6. d Box-and-Whisker plot of relative XPO1 expression in cervical cancer cell lines (n = 9), normal tissues (n = 24), and cervical cancer tissues (n = 28) (GDS3233). e Immunoblot analysis of HP1? immunoprecipitates (IP) and whole-cell lysates (WCL) from C33A cells expressing Flag-HPV16 E6.