Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) from HEK293T cells transfected with GFP-Ub and Flag-YTHDF1. b YTHDF1 polyubiquitination could largely be detected in cells transfected with indicated constructs. c IB analysis of WCL and His immunoprecipitate from HEK293T cells transfected with indicated constructs. d PLC/PRF/5 cells were immunoprecipitated with either anti-USP5 or anti-YTHDF1 antibody and then analyzed by IB. e IB and IP products analysis of USP5-YTHDF1 interaction in HEK293T cells expressing HA-USP5 WT or the indicated truncated YTHDF1 mutants. f , g IB analysis of YTHDF1 levels in HEK293T cells expressing HA-USP5 (DNA content of 250 ng or 500 ng) or indicated plasmids. h IB and QRT-PCR analysis of YTHDF1 from Hepa1-6 cells with Usp5 knockout. n = 3. i , j IB analysis of WCL from Hepa1-6 cells with the depletion of Usp5 or HEK293T cells transfected with indicated constructs for 36 h. Cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 levels was quantified by the ImageJ software. k IB analysis of WCL and IP products from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. l Effects of Usp5 knockout in Hepa1-6 cells on Ythdf1 K11-linked polyubiquitination were evaluated by IB. m Effects of WP1130 on USP5-mediated YTHDF1 K11-linked polyubiquitination. Cells expressing indicated plasmids were treated with different doses of WP1130. n IB analysis of proteins labeled with puromycin using anti-puromycin antibody upon Usp5 depletion with or without expressing Ythdf1. o , p Assessment of subcutaneous tumor formation from PLC/PRF/5 cells after depletion of USP5, or YTHDF1, or stably expressing YTHDF1 with endogenous USP5 knockdown. Tumor weight was measured at the endpoint of the study. Tumor growth was measured at the indicated time points. n = 5. * p < 0.05, t -test. q Kaplan-Meier analysis revealed a relationship between YTHDF1 and USP5 expression and overall survival in HCC patients. All data are presented as mean ± SEM. All IB data are representative of three independent experiments. Source data are provided as a file.

Article Snippet: For drug treatments, MG132 (S2619), WP1130 (S2243), AKTi-1/2 (S7776), Rapamycin (S1039), Wortmannin (S2758), and Torin1 (S2827) were purchased from Selleck Chemicals.

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Expressing, Quantitative RT-PCR, Knock-Out, Software, Labeling, Stable Transfection, Knockdown

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Kaplan-Meier analysis of USP5 and CD4 or CD8 expression and overall survival of 90 HCC patients. b A total of 5.0 × 10 6 WT or Usp5-KO Hepa1-6 cells were subcutaneously implanted in C57BL/6 mice pretreated with anti-IgG, anti-NK1.1, anti-CD4, and anti-CD8 antibodies (n = 6). Tumor growth was monitored at the indicated time points. *** p < 0.001 by two-way ANOVA. c IB analysis of lysates derived from dissected xenografts formed by Usp5-depleted H22 or Hepa1-6 cells. d , e IB analysis of Pd-l1 in the WT and Usp5-KO Hepa1-6 cells transfected with Flag-Ythdf1 constructs for 36 h or treated with 2.5 μM WP1130 for 24 h. f Activated T cells and tumor cells were co-cultured in 24-well plates for 4 days and surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown. n = 3. ** p < 0.01. t- test. g Correlation between the fold changes of differentially expressed genes in Ythdf1 and Usp5 knockout Hepa1-6 cells. h The top 5 terms in GO analysis of the immune-related genes suppressed by both Usp5 and Ythdf1 knockout Hepa1-6 cells. n = 3. i GSEA and heatmap showing the differential expression of genes in Fig. 5g. n = 3. j mRNA levels of indicated genes from sgUsp5 or sgCtrl Hepa1-6 cells were analyzed using QRT-PCR. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. t- test. k The mRNA and protein levels in Usp5 and Ythdf1 knockout Hepa1-6 cells are measured by RNA-Seq and proteomics. l , m Usp5 or Ythdf1 depletion affects tumor growth in mouse xenograft in NOD/SCID and C57BL/6 mice ( n = 8). Tumor growth was monitored at the indicated time points, and tumor volume were measured at the endpoint ( l ). Tumor image and tumor weight are presented ( m ). *** p < 0.001 by two-way ANOVA. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: For drug treatments, MG132 (S2619), WP1130 (S2243), AKTi-1/2 (S7776), Rapamycin (S1039), Wortmannin (S2758), and Torin1 (S2827) were purchased from Selleck Chemicals.

Techniques: Expressing, Derivative Assay, Transfection, Construct, Cell Culture, Staining, Knock-Out, Quantitative Proteomics, Quantitative RT-PCR, RNA Sequencing

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Tumor growth of sgCtrl and sgUsp5 Hepa1-6 cells in C57BL/6 mice with anti-IgG mAb or anti-PD-L1 mAb treatments. n = 10. b Kaplan-Meier survival curves for four treatment groups demonstrate the improved efficacy of PD-L1 mAb after knockout Usp5. ** P < 0.01. c A schematic model illustrating the treatment plan for mice bearing subcutaneous Hepa1-6 or H22 tumors. Male C57BL/6 mice were implanted with Hepa1-6 or H22 cells subcutaneously and treated with four arms: control antibody (anti-IgG mAb) treatment, anti-PD-L1 mAb treatment, USP5 inhibitor WP1130 treatment, and anti-PD-L1 mAb plus USP5 inhibitor combination treatment. d Hepa1-6 and H22 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody, anti-PD-L1 mAb, the USP5 inhibitor WP1130, or combined therapy were measured every 3 days and plotted individually. n = 10 mice per group. e Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-L1 mAb with the USP5 inhibitor WP1130. *** P < 0.001. f Immunohistochemical (IHC) analysis of Cd8, Granzyme B (GzmB), Tim-3, Ythdf1 and Pd-l1 expression in Hepa1-6 tumors after indicated treatments. g Representative multiplex immunohistochemistry (mIHC) images of Cd8 (Gray), GzmB (Green), Tim-3 (Red), and DAPI nuclear staining (blue) in Hepa1-6 tumors after indicated treatments. All data are presented as mean ± SEM. Source data are provided as a file.

Article Snippet: For drug treatments, MG132 (S2619), WP1130 (S2243), AKTi-1/2 (S7776), Rapamycin (S1039), Wortmannin (S2758), and Torin1 (S2827) were purchased from Selleck Chemicals.

Techniques: Knock-Out, Control, Immunohistochemical staining, Expressing, Multiplex Assay, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: Representative USP5 protein staining of tumor sections (top: 100×, bottom: 400×) (left) and CT scans (right) from liver cancer patients ( a ) or lung cancer patients ( b ) undergoing anti-PD-1 treatment. CT scans of patient tumors are shown. c , d Waterfall plot depicting the responses to anti-PD-1 treatment based on the best change in the sum of target lesions compared to baseline in cancer patients with low USP5 or high USP5 expression. Each bar represents one patient, and the colors correspond to the response to anti-PD-1 treatment (PR: partial response, SD: stable disease, PD: progressive disease). Dotted black lines indicate the response as described by RECIST1.1. e , f Pie charts illustrating the response fractions for each group of patients with USP5-low and USP5-high expression in tumor cells. g IB analysis of indicated proteins derived from two fresh HCC samples (HCC180328 and HCC191017) before the establishment of the humanized mouse model. h IB analysis of indicated proteins in a subcutaneous humanized mouse model after 21 days of treatment with anti-IgG or anti-PD-1. i A working model for targeting USP5 to sensitize tumors to PD-1/PD-L1 blockade. USP5 downregulates PD-L1 and enhances the immunosuppressive response, leading to resistance to PD-1/PD-L1 blockade (Left Panel). Inhibition of USP5 by WP1130 upregulates PD-L1 and reduces the immunosuppressive response to sensitize the tumor to anti-PD-1/PD-L1 immunotherapy (Right Panel). All IB data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: For drug treatments, MG132 (S2619), WP1130 (S2243), AKTi-1/2 (S7776), Rapamycin (S1039), Wortmannin (S2758), and Torin1 (S2827) were purchased from Selleck Chemicals.

Techniques: Staining, Expressing, Derivative Assay, Inhibition