Figure 4: UVRAG^FS promotes chromosomal instability and centrosome amplification.

(a) Representative SKY analysis of mouse embryonic stem (ES) cells expressing vector or Flag-UVRAG^FS. Average chromosomal aberrations per cell were quantified. *P<0.05, Wilcoxon Signed-rank Test. (b) Chromosomal rearrangement and single-nucleotide variants (SNVs) in UVRAG^FS gastric cancer. Representative Circos plots of the UVRAG^WT and UVRAG^FS subtypes of MSI gastric cancer in the Pfizer and UHK cohorts²⁰. The inner circle denotes chromosomal structural variants: ITX (intrachromosomal translocation), blue; DEL (deletion), red; CTX (interchromosomal translocation), green; INV (inversions), pink; Tandem duplication, black. In the second circle, each dot denotes one somatic SNV, coloured according to six mutation types: T>G, pink; T>C, green; T>A, gray; C>G, black; C>A, blue; C>T, red. The outer circle denotes 23 chromosomes. Boxplot represents the total chromosomal structure variants (SV) count in the UVRAG^WT and UVRAG^FS MSI gastric cancers (Mann–Whitney test). (c) UVRAG^FS induces centrosome amplification. SW480.Vector and SW480.UVRAG^FS cells with different centrosome numbers were immunostained for γ-Tubulin and DAPI and quantified (Data are the means±s.d., n=200 cells obtained by gathering data from three independent experiments). (c) centrosome. Scale bar, 10 μm. (d) Representative confocal images of spindle malformation in mitotic Vector, UVRAG^WT, and UVRAG^FS SW480 cells co-stained with anti-γ-Tubulin (red) and anti-α-Tubulin (green) for the mitotic asters. The percentages of cells with disorganized spindle were quantified. Data are the means±s.d. (n=200 cells obtained by pooling data from three independent experiments). Scale bar, 10 μm. (e) UVRAG^FS is defective in CEP63 binding. Whole-cell lysates (WCL) of 293T transfected with Flag-UVRAG^WT or Flag-UVRAG^FS were immunoprecipitated with anti-Flag followed by IB with anti-CEP63. (f) Representative image showing dissociation of UVRAG^FS from the centrosome. HeLa cells expressing Flag-UVRAG^WT or Flag-UVRAG^FS were stained with anti-Flag (green), anti-γ-Tubulin (red), and anti-CEP63 (blue). Scale bar, 10 μm. (g) UVRAG^FS inhibits UVRAG-CEP63 interaction. The 293T cells were transfected with increasing amounts of Flag-UVRAG^FS. WCL were immunoprecipitated with anti-CEP63 or anti-Flag, followed by immunoblotting with anti-UVRAG or anti-CEP63 as indicated.