Fig. 1: USP1 is an important regulator of Hippo signaling activity, and its expression is elevated in human hepatocellular carcinoma.

A The flowchart shows the siRNA screening procedure for identifying novel deubiquitinases involved in modulating Hippo signaling. HEK293 cells were seeded in 24-well plates. Each well was transfected with a siRNA against one deubiquitinase and incubated for 48 h. RNA was extracted, and CTGF expression was assessed by qPCR. B In the siRNA screen of deubiquitinases, relative CTGF mRNA levels were determined. The red columns represent relative mRNA level of CTGF after depletion of USP1. C The relative RNA level of USP1 in HCC tumor samples (n = 371) versus normal samples (n = 50) in the TCGA database (https://portal.gdc.cancer.gov/). D The relative RNA level of USP1 in HCC tumors of different stages (Stag I, n = 168; Stag II, n = 84; Stag III, n = 82; Stag IV, n = 6) was compared with that in normal liver tissue (n = 50). Data were obtained from the TCGA database (https://portal.gdc.cancer.gov/). E Comparing with lower USP1 expression (n = 275), higher USP1 expression (n = 90) was associated with poorer overall survival in HCC patients based on Kaplan‒Meier analysis in TCGA. P < 0.001, log-rank test. F TCGA analysis of the correlation of USP1 expression with that of classical downstream genes of the Hippo signaling pathway in HCC (n = 371), with p < 0.001 as the threshold. G USP1 expression was significantly correlated with that of CCN1 (CYR61) and CCN2 (CTGF) in HCC (n = 371). H Volcano plot of RNA-seq data in HLF cell lines treated with siControl or siUSP1. The volcano plot revealed a significant increase in the expression of CTGF and CYR61 downstream of YAP as a result of USP1 depletion. Threshold values of P < 0.05 and fold change>2 was set as screening criteria. I These are the top 10 KEGG pathways that are significantly depleted (top) or enriched (bottom) in HLF cells treated with siUSP1. The pathway enrichment analysis consisted of differentially regulated genes identified with threshold criteria of P < 0.001 and fold change >2. The cells were treated for 48 h with 50 nM siUSP1 or vehicle. Total mRNA was extracted for RNA sequencing analysis. n = 3. J HLF cells treated with USP1 siRNA had depletion of Hippo pathway signature genes, according to gene set enrichment analysis (GSEA).