Figure 1: Identification of UVRAG FS mutation in CRC cell lines and primary tumours.

(a) Sequencing analysis of UVRAG at the location of the A₁₀ repeat in MSS (HCC2998, COLO205, SW620, SW480 and HT29) and MSI (HCT15, SW48, HCT116, RKO, LIM2405, LS180 and KM12) CRC cell lines. Arrows indicate the heterozygous deletion of one A in UVRAG A₁₀ in MSI cell lines. (b,c) Wild-type (WT) and FS mutant UVRAG protein expression in MSS and MSI CRC cell lines. Whole-cell lysates (WCL) of MSS and MSI CRC cell lines were immunoprecipitated with anti-UVRAG^FS followed by immunoblotting with anti-UVRAG^FS, or they were directly probed with antibodies targeting UVRAG^WT or γ-H2AX. Actin served as a loading control. Densitometric quantification of protein expression is shown in (c). Dash lines indicate average band intensities of all the tested cell lines. Note reduced UVRAG^WT expression in MSI CRC cells expressing UVRAG^FS. (d) H&E (first row) and immunohistochemical analysis of UVRAG (second row), Ki67 (fourth row), and γ-H2AX (5th row) in paired human primary CRC specimen obtained from three separate patients with their corresponding status of UVRAG FS mutation (third row) provided. The bar plots (right) are the quantification of the levels of Ki67 and γ-H2AX (denoted by arrows) in the paired tissues with WT or mutant UVRAG. HPF, high-power field. ***P<0.001 (Mann–Whitney test); Scale bar ,50 μm.