Fig. 4

USP5 promotes Wnt/β-catenin signaling by deubiquitinating and stabilizing β-catenin. A Significant correlations between USP5 expression and ssGSEA scores of the REACTOME_SIGNALING_BY_WNT and REACTOME_TCF_DEPENDENT_ SIGNALING_IN_RESPONSE_TO_WNT gene sets in the TCGA-LUAD dataset. r, Pearson correlation coefficient. B–D Protein levels of USP5 and β-catenin in CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells (B), A549 cells stably expressing a vector or USP5 (C), and CL1-5 cells stably expressing a vector or USP5 isoform #1 or #2 under Wnt3a treatment (D). β-actin was used as an internal control. E CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells treated with different concentration of Wnt3A and tested to measure the protein levels of USP5 and β-catenin. F Immunohistochemical staining for USP5 and β-catenin in CL1-5-shLacZ and shUSP5#2 tumor samples. Scale bar: 50 μm. G and H CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells treated with 20 µg/ml protein synthesis inhibitor cycloheximide (CHX) for 60 min. The indicated proteins were detected by western blotting (G), and the results were quantified with ImageJ software (H). **P < 0.01 by a two-tailed Student’s t test. I Immunoprecipitation of endogenous USP5 in CL1-5 cells with antibodies against USP5 or an IgG control antibody. The associated β-catenin protein was detected with an anti-β-catenin antibody. J Immunoprecipitation of endogenous β-catenin in CL1-5 cells with antibodies against β-catenin or an IgG control antibody. The associated USP5 protein was detected with an anti-USP5 antibody. K CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells treated with 10 µM MG132 for 6 h, followed by immunoprecipitation of ubiquitinated β-catenin proteins under denaturing conditions with anti-β-catenin antibodies. The ubiquitination of β-catenin was detected by western blotting using an anti-ubiquitin antibody. L The mRNA levels of Wnt target genes in CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells. **P < 0.01 and ***P < 0.001 by a two-tailed Student’s t test. M A549 cells stably expressing either pLEX or pLEX-USP5 was used for silencing β-catenin expression, and each group was cultured at 1,000 cells per well in low-attachment plates to assess sphere formation. Left panel, representative stitched brightfield images were produced using NIS-Elements software (Nikon). Scale bar: 100 μm. Right panel, quantification of sphere formation after 14 days. *P < 0.05, **P < 0.01 by a two-tailed Student’s t test