FIGURE 1.

Ubiquitin‐specific protease 28 (USP28) is important for Wnt signaling in hepatocellular carcinoma cells. (A) Expression of USP28 in liver cancer samples and normal tissues (paracancer). Three hundred and fifty‐one of the samples were cancerous and 61 were paracancer. (B) Distribution of USP28 expression in different pathological stages of liver cancer. WILCOX test was used to calculate the differences among the three groups and p values were directly calculated. (C) Relationship between USP28 expression and survival in patients with liver cancer. Software X‐tile was used to obtain the value of cut‐off. Cut‐off (USP28) = 1.8068. All data are from The Cancer Genome Atlas database with 351 data in (A–C). (D) Correlation between USP28 and Wnt signaling pathway was examined using Gene Set Enrichment Analysis. (E) TOPFlash reporter assay in HepG2 and HuH6 cells depleted of USP28 by two independent shRNAs. Results are expressed as relative (TOP/Renilla)/(FOP/Renilla) luciferase activity. (F, G) Western blot analysis of USP28 and the cell viability detection in HepG2 (F) and HuH6 (G) cells with USP28 depleted by shRNA‐mediated knockdown. Cell viability was determined using MTS assay and the absorbance was 490 nm. (H) Ability for plate clone formation in HepG2 and HuH6 cells depleted of USP28 by two independent shRNAs. (I) Western blot analysis of USP28 in HepG2 cells depleted of USP28 by two independent shRNAs followed the exogenous overexpression of USP28. (J) Cell viability of HepG2 depleted of USP28 by two independent shRNAs followed the exogenous overexpression of USP28 determined using MTS assay. All data are representative of three independent experiments (mean ± SEM). Two‐tailed Student's t‐tests were applied to examine statistical significance. **p < 0.01; ***p < 0.001; ****p < 0.0001.