Additional file 1 of Ubiquitin-specific peptidase 5 facilitates cancer stem cell-like properties in lung cancer by deubiquitinating β-catenin
Creators: name: Tung, Chia-Hao
name: Wu, Jia-En
name: Huang, Meng-Fan
name: Wang, Wen-Lung
name: Wu, Yi-Ying
name: Tsai, Yao-Tsung
name: Hsu, Xiu-Rui
name: Lin, Sheng-Hsiang
name: Chen, Yuh-Ling
name: Hong, Tse-Ming
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. Sequences of specific paired primers used in this study. Figure S1. Deubiquitination-related Pathway is positively correlated with stemness and poor overall survival in TCGA-LUAD dataset. a Heat map showing 135 reactome pathways positively correlated with stemness index mRNAsi in TCGA-LUAD dataset. The ssGSEA score of each pathway was determined using R/Bioconductor package GSVA (v1.34.0). Correlation between pathway ssGSEA score and mRNAsi was determined using Pearson correlation analysis. Pathways with the correlation coefficients larger than 0.7 were selected for the positively correlated pathways. b Univariate Cox regression analysis of ssGSEA pathway scores for overall survival in TCGA-LUAD dataset. Forest plot showing the top 10 pathways (in the 135 stemness-associated pathways) associated with poor overall survival in TCGA-LUAD dataset. HR, hazard ratios; CI, confidence intervals. Figure S2. USP5 is associated with mRNAsi and clinical outcomes in human lung cancer. a Heat map showing the correlation between 7 DUBs and stemness index mRNAsi in TCGA-LUAD dataset. Correlation was determined using Pearson correlation analysis. b, c The expression levels of USP5 in lung tumor samples (n = 91) were significantly higher than normal lung tissues (n = 65) in both GSE19188 (b) and GSE2514 © dataset. d The expression levels of USP5 are significantly higher in late-stage tumors (Stage III, IV; n = 26), compared to early-stage tumors (Stage I, II; n = 85) in GSE3141 dataset. e USP5 expression in lung cancer patients with lymph node metastasis (N1; n = 52) are significantly higher than those without metastasis (N0; n = 129) in GSE50081 dataset. P-value was determined by Mann-Whitney U test. Figure S3. USP5 promotes sphere formation in lung cancer. a A549 cells stably expressing either pLEX or pLEX-USP5 was cultured at 5,000 cells per well in low-attachment plates to assess sphere formation. Representative stitched brightfield images were produced using NIS-Elements software (Nikon). Scale bar: 200 μm. b Quantification of spheres generated by A549 cells stably expressing either pLEX or pLEXUSP5 after 14 days. *P &lt; 0.05 by a two-tailed Student’s t test. Figure S4. USP5 correlates with SNAI2 expression and EMT in lung cancer. A Correlation between stemness index mRNAsi and ssGSEA score of gene set SARRIO_EMT_UP in TCGA-LUAD dataset, determined using Pearson’s correlation analysis. b The mRNA levels of SNAI2 in CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells. ***P &lt; 0.001 by two-tailed Student’s t-test. c, d The relationships between USP5and SNAI2 were determined from gene expression data from GSE50081 (c) and GSE17710 (d) datasets using Pearson’s correlation analysis. Figure S5. Knockdown of USP5 suppresses β-catenin expression in lung cancer cells. a Gene set enrichment analysis reveals USP5 expression is positively correlated with Wnt signaling pathways using TCGA-LUAD dataset. Black bars at the bottom of the figure indicate the location of genes in each gene set. b The mRNA levels of CTNNB1and GAPDH in CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells. c The protein levels of USP5 and β-catenin in control (shLacZ) and USP5-depleted (shUSP5) lung cancer cells. β-actin was used as an internal control. d The protein levels of USP5 and β-catenin in USP5-overexpressing and control A549 cells with or without β-catenin knockdown. Figure S6. Targeting USP5 via small compounds suppresses β-catenin expression and enhances its ubiquitination in lung cancer cells. a The protein levels of USP5 and β-catenin in LIJ cells treating with either DMSO control or WP1130. b DMSO- or WP1130-treated LIJ cells were treated with 10 μM MG132 for 6 h and pulled down under denaturing conditions using anti-β-catenin antibodies. The ubiquitinated β-catenin was detected by western blotting using an anti-ubiquitin antibody. c The protein levels of USP5 and β-catenin in CL1-5 cells treating with either DMSO control or Vialinin A. β-actin was used as an internal control. Figure S7. Kaplan-Meier survival curve of lung cancer patients based on the combined markers of USP5, Wnt signaling, and stemness scores. The global gene expression data was obtained from 293 lung cancer patients in the Rousseaux_2013 dataset. The ssGSEA scores of Wnt signaling and stemness were calculated by R/Bioconductor package GSVA (v1.34.0) using gene sets of REACTOME_TCF_DEPENDENT_SIGNALING_IN_RESPONSE_TO_WNT and BENPORATH_ES_CORE_NINE, respectively. a The patients were divided into the high- and lowexpression of each factor using the median value as the cutoff. The combined effects of USP5, Wnt signaling, and stemness on the overall survival of lung cancer patients were analyzed. The median survival of each molecular subtype is indicated. **P &lt; 0.01, *P&lt; 0.05 by log-rank test. b Relative proportions of patients categorized based on USP5 expression, Wnt signaling score, and stemness score.