Fig 2: PICK1 inhibits proliferation of NPC cells in vitro.A, B The viability of the indicated cells was examined using the CCK-8 assay. C–H The indicated cell proliferation was determined via colony formation assay (C, D) and EdU Incorporation Assay (E–H). Representative images and quantitative analyses are shown. The data are displayed as the means ± SD of three independent tests. ***p < 0.001, **p < 0.01, *p < 0.05.

Fig 3: PICK1 inhibits the Wnt/β-catenin signaling pathway.A, B Western blotting assay was used to examine the expression levels of β-catenin, c-Myc, and cyclinD1 in NPC cells after PICK1 knockdown or overexpression. Actin served as the loading control. C, D Immunohistochemistry analysis showing the expression of PICK1 and β-catenin in mouse tumor tissues. E, F PICK1 and β-catenin protein expression in mouse tumor tissues was examined by western blotting.

Fig 4: PICK1 accelerates proteolytic degradation of β-catenin.A–E Western blot analysis of β-catenin protein levels in NPC cells stably knocking down or overexpressing PICK1 treated with CHX (A–D) and MG132 (E). GAPDH acted as the loading control. F 293 T cells transfected with the indicated siRNA or plasmids were treated with MG132 (10 μM) for 6 h before collection. Ubiquitylation assays were conducted and the ubiquitylation level of β-catenin was analyzed using an anti-His antibody. G Western blot analysis of pGSK-3β and β-catenin expression in NPC cells with stably silenced PICK1 or in which PICK1 was stably overexpressed. GAPDH served as the loading control. H Western blot analysis of pGSK-3β and β-catenin levels in NPC cells with vector or Flag-PICK1, followed by treatment with or without LiCl (20 mM) for 24 h. Actin served as the loading control.

Fig 5: PICK1 promotes β-catenin nuclear accumulation.A, B Subcellular β-catenin localization in NPC cells was further evaluated by immunofluorescence. C, D Nuclear and cytoplasmic β-catenin protein levels in NPC cells with overexpressed or silenced PICK1 were measured by western blotting. Internal loading controls used LaminB1 and α-Tubulin. E, F RT-qPCR was used to assess the mRNA levels of β-catenin in the indicated cells.