Fig 2: NUSAP1 contributes to the activation of canonical Wnt/β-catenin signaling. a GSEA plot showing that NUSAP1 expression positively correlates with Wnt/β-catenin-activated gene signatures (LABBE_WNT3A_TARGETS_UP). b TOP/FOP luciferase assay of TCF/LEF transcriptional activity in the indicated cells. c Western blotting analysis of β-catenin expression in the cytoplasm and nucleus of the indicated cells. GADPH and p84 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. d Western blotting analysis of the protein expression levels of the β-catenin target genes in the indicated cells. GADPH was used as the loading control. e Lysates from Siha cells transfected with HA-TCF4 and Flag-SUMO1 were immunoprecipitated with anti-HA affinity agarose, followed by western blot analysis of TCF4 and SUMO1. f, g Lysates of Siha cells were immunoprecipitated using NUSAP1 or RanBP2 antibodies, and the interactions of NUSAP1 with TCF4 and RanBP2 were analysed by immunoblotting. h Siha cells were transfected with the indicated control RNA and siRNA against RanBP2. Lysates were immunoprecipitated with anti-HA antibody, followed by western blot analysis of TCF4 and SUMO1. i. TOP/FOPluciferase assay of TCF/LEF transcriptional activity in the NUSAP1-ovexxpressed Siha cells transfected with the indicated control RNA and SiRNA against RanBP2.All experiments were performed in triplicate. Results are the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 3: Silencing PLPP4 inhibits Ca2+-permeable cationic channel in lung carcinoma cells. a FACS analysis showed that silencing PLPP4 decreased intracellular Ca2+ in the indicated cells. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. b Western blot analysis of cytoplasmic and nuclear expression of the S54 phosphorylated NFAT1 in the indicated cells. α-Tubulin was used as the loading control and the nuclear protein p84 was used as the nuclear protein marker. c Relative NFAT reporter activity in the indicated cells. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. d Hypothetical model illustrating that activation of Ca2+-permeable cationic channel by PLPP4 contributes to proliferation and cell cycle progression in lung cancer cells

Fig 4: Canonical Wnt/β-catenin signaling mediates the effects of NUSAP1 and clinical relevance of NUSAP1-induced β-catenin activation in cervical cancer. a Western blotting analysis confirming the depletion of β-catenin in NUSAP1-transduced cells using a β-catenin-siRNA or XAV-939. b TOP/FOP luciferase activity in the indicated cells (c) Quantification of the invading cells in a matrigel-coated transwell assay. d, e Real-time PCR analysis of EMT markers such as E-cadherin (d) and vimentin (e) in the indicated cells. f Histograms showing the mean number of spheres formed by the indicated cells. g, h. Western blotting analysis of NUSAP1 and nuclear β-catenin protein levels in ten fresh cervical cancer tissues. GADPH and p84 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. Right panel, correlation analyses between the protein expression of NUSAP and nuclear β-catenin in ten fresh cervical cancer samples. i, j. Immunohistochemical analysis of NUSAP1 and nuclear β-catenin protein levels in cervical cancer tissues. Left panel, images of two representative cases. Right panel, percentages of cervical cancer specimens with low or high expression of NUSAP1 relative to nuclear β-catenin expression levels. Results are the mean ± SD. **p < 0.01; ***p < 0.001