Fig 2: USP5 is overexpressed and highly associated poor prognosis in NSCLC patients.A Copy number analysis of USP5 in TCGA lung adenocarcinoma. B Kaplan–Meier survival plot of USP5 mRNA in lung cancers using KM plotter (https://kmplot.com/). C IHC analysis of PD-L1 and USP5 protein levels in NSCLC tissues. The correlation between USP5 and PD-L1 in NSCLC patient tumors (n = 112) was explored using Pearson correlationanalysis. D IHC staining of USP5 was compared in NSCLC tissues and normal lung tissues. E Kaplan–Meier survival curve of USP5 protein expression in NSCLC patients, n = 74.

Fig 3: PD-L1 is physically associated with USP5.A Silver staining and mass spectrometry analysis of PD-L1 immunoprecipitation complex in H1299 cells. Representative peptides and coverage of PD-L1 and USP5 are shown. B, C Co-immunoprecipitation (co-IP) analysis from lysates derived from H1299 and H460 cells using anti-PD-L1 (B) or anti-USP5 (C) antibodies. D The coomassie blue staining of purified recombinant GST tagged USP5 and His tagged PD-L1 proteins. E In vitroGST-pull down analysis was performed using GST-USP5 and His-PD-L1.

Fig 4: USP5 knockdown inhibits tumor growth in Lewis lung cancer xenografts.A Western blotting analysis of Lewis lung carcinoma (LLC) cells expressing control (ctrl) or USP5 shRNA. B, C LLC cells expressing ctrl or USP5 shRNA have been implanted into C57/BL6 mice, B Tumor growth curve and C tumor weights have been measured. D Immunohistochemistry (IHC) analysis of USP5, PD-L1, and Ki67 levels in LLC xenografts expressing ctrl or USP5 shRNA. Representative image (left) and quantification (right) of IHC stainig were shown. E IHC staining analysis of CD8 and Grazyme B positive cells in LLC xenografts expressing ctrl or USP5 shRNA. Data represents the mean ± SD, n = 5 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: USP5 deubiquitinates PD-L1.A Immunoprecipitates and western blot analysis of cell lysates derived from H1299 cells transfected with HA-Ub, Flag-PD-L1, Myc-USP5 WT/C335A and treated with 5 µM MG132 for 6 h. B H1299 cells with or without USP5 knockdown by siRNA were co-transfected with HA-Ub and Flag-PD-L1, immunoprecipitation was performed using anti-FLAG affinity beads after 5 µM MG132 treatment for 6 h. Poly-ubiquitination of PD-L1 was analyzed by western blot with anti-HA antibodies. C H1299 cells were transfected with or without USP5 siRNA, 72 h after transfection, cells were lysed and subjected to anti-PD-L1 immunoprecipitation (IP), followed by analysis of PD-L1 ubiquitination using immunoblotting with anti-Ub(ubiquitin) antibody. D USP5 deubiquitinates polyubiquitination of PD-L1 in vitro. Flag-PD-L1 and HA-Ub were transfected in HEK293T cells. Poly-ubiquitinated PD-L1 was then immunoprecipitated with anti-Flag M2 beads and incubated with purified USP5-WT/C335A protein as indicated time. Lysates were analyzed by IB with indicated antibodies.