Journal: PLOS One

Article Title: An in vitro tumor recurrence model based on platinum-resistant colon cancer cells as a research tool for studying cancer cell dormancy

doi: 10.1371/journal.pone.0333671

Figure Lengend Snippet: (A,B) LysoTracker-positive acidic lysosomal compartments in quiescent cells. Oxaliplatin-resistant HCT116 oxpl-R cells (A) and cisplatin-resistant HCT116 cspl-R cells (B) before (day 0) and after (day 8) platinum treatment (oxaliplatin or cisplatin, respectively), stained with LysoTracker Green™ and MitoTracker Orange™. Scale bars represent 50 µm. (C,D) Dynamics of autophagy marker expression in the in vitro cancer reсurrence model based on cisplatin-resistant colon cancer cells HCT116 cspl-R, analyzed by immunoblotting (C) and qPCR (D) . (C) Immunoblots probed with LC3, Beclin and Survivin antibodies. α-Tubulin was used as a loading control. Quantification values shown beneath each lane represent band intensity normalized to loading control and relative to day 0 control (set as 1.0). (D) Normalized expression of autophagy-related genes ( lc3, beclin ) after cisplatin exposure (days 0-33), relative to day 0. Expression of the GAPDH gene served as the endogenous control. Data represent biological triplicate experiments and are displayed as mean ± SEM.

Article Snippet: The primary antibodies used were against LC3 #4108 RRID: AB_2137703, Beclin #3738 RRID:AB_490837, Survivin #2808 RRID:AB_2063948 (Cell Signaling, USA), Cyclin A sc-751 RRID:AB_631329, and p27 (C-19) sc-528 RRID:AB_632129 (Santa Cruz Biotechnology, USA) with ɑ-Tubulin sc-32293 RRID:AB_628412 (Santa Cruz Biotechnology, USA) serving as a loading control.

Techniques: Staining, Marker, Expressing, In Vitro, Western Blot, Control

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: Anti-HA-Tag (1:1000 dilution, Cat# 3724, RRID:AB_1549585), anti-GFP-Tag (1:1000 dilution, Cat# 2956, RRID:AB_1196615), anti-T7-Tag (1:1000 dilution, Cat# 13246, RRID:AB_2798161), anti-Myc-Tag (1:1000 dilution, Cat# 2278, RRID:AB_490778), anti-His-Tag (1:1000 dilution, Cat# 12698, RRID:AB_2744546), anti-ubiquitin (1:1000 dilution, Cat# 3936, RRID:AB_331292), anti-PD-L1 (1:1000 dilution, Cat# 13684, RRID:AB_2687655), anti-YTHDF1 (1:1000 dilution, Cat# 86463, RRID:AB_2923055), anti-YTHDF2 (1:1000 dilution, Cat# 80014 RRID:AB_2923056), anti-mTOR (1:1000 dilution, Cat# 2983, RRID:AB_2105622), anti-Phospho-mTOR (Ser2448) (1:1000 dilution, Cat# 5536, RRID:AB_10691552), anti-4E-BP1 (1:1000 dilution, Cat# 9644, RRID:AB_2097841), anti-Phospho-4E-BP1 (Thr37/46) (1:1000 dilution, Cat# 2855, RRID:AB_560835), anti-p70 S6 (1:1000 dilution, Cat# 9202, RRID:AB_331676), anti-Phospho-p70 S6 (Thr389) (1:1000 dilution, Cat# 97596, RRID:AB_2800283), anti-Acetyl-CoA Carboxylase 1 (1:1000 dilution, Cat# 4190, RRID:AB_10547752), anti-Phospho-Acetyl-CoA Carboxylase (Ser79) (1:1000 dilution, Cat# 11818, RRID:AB_2687505), anti-AKT (1:1000 dilution, Cat# 4691, RRID:AB_915783), anti-Phospho-Akt (Ser473) (1:1000 dilution, Cat# 4060, RRID:AB_2315049), anti-SIN (1:1000 dilution, Cat# 12860, RRID:AB_2798048), anti-GβL (1:1000 dilution, Cat# 3274, RRID:AB_823685), anti-Raptor (1:1000 dilution, Cat# 2280, RRID:AB_561245), anti-RICTOR (1:1000 dilution, Cat# 2114, RRID:AB_2179963), anti-DEPTOR (1:1000 dilution, Cat# 11816, RRID:AB_2750575), anti-DYKDDDDK (1:1000 dilution, Cat# 14793, RRID:AB_2572291), anti-USP1 (1:1000 dilution, Cat# 8033, RRID:AB_10858879), anti-USP15 (1:1000 dilution, Cat# 66310, RRID:AB_2799706), Rabbit (DA1E) mAb IgG XP® Isotype Control (Cat# 3900, RRID:AB_1550038), Mouse (G3A1) mAb IgG1 Isotype Control (Cat# 5415, RRID:AB_10829607), anti-Lamin B1 (1:1000 dilution, Cat# 13435, RRID:AB_2737428), anti-GAPDH (1:1000 dilution, Cat# 2118, RRID:AB_561053) were purchased from Cell Signaling Technology.

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: Anti-HA-Tag (1:1000 dilution, Cat# 3724, RRID:AB_1549585), anti-GFP-Tag (1:1000 dilution, Cat# 2956, RRID:AB_1196615), anti-T7-Tag (1:1000 dilution, Cat# 13246, RRID:AB_2798161), anti-Myc-Tag (1:1000 dilution, Cat# 2278, RRID:AB_490778), anti-His-Tag (1:1000 dilution, Cat# 12698, RRID:AB_2744546), anti-ubiquitin (1:1000 dilution, Cat# 3936, RRID:AB_331292), anti-PD-L1 (1:1000 dilution, Cat# 13684, RRID:AB_2687655), anti-YTHDF1 (1:1000 dilution, Cat# 86463, RRID:AB_2923055), anti-YTHDF2 (1:1000 dilution, Cat# 80014 RRID:AB_2923056), anti-mTOR (1:1000 dilution, Cat# 2983, RRID:AB_2105622), anti-Phospho-mTOR (Ser2448) (1:1000 dilution, Cat# 5536, RRID:AB_10691552), anti-4E-BP1 (1:1000 dilution, Cat# 9644, RRID:AB_2097841), anti-Phospho-4E-BP1 (Thr37/46) (1:1000 dilution, Cat# 2855, RRID:AB_560835), anti-p70 S6 (1:1000 dilution, Cat# 9202, RRID:AB_331676), anti-Phospho-p70 S6 (Thr389) (1:1000 dilution, Cat# 97596, RRID:AB_2800283), anti-Acetyl-CoA Carboxylase 1 (1:1000 dilution, Cat# 4190, RRID:AB_10547752), anti-Phospho-Acetyl-CoA Carboxylase (Ser79) (1:1000 dilution, Cat# 11818, RRID:AB_2687505), anti-AKT (1:1000 dilution, Cat# 4691, RRID:AB_915783), anti-Phospho-Akt (Ser473) (1:1000 dilution, Cat# 4060, RRID:AB_2315049), anti-SIN (1:1000 dilution, Cat# 12860, RRID:AB_2798048), anti-GβL (1:1000 dilution, Cat# 3274, RRID:AB_823685), anti-Raptor (1:1000 dilution, Cat# 2280, RRID:AB_561245), anti-RICTOR (1:1000 dilution, Cat# 2114, RRID:AB_2179963), anti-DEPTOR (1:1000 dilution, Cat# 11816, RRID:AB_2750575), anti-DYKDDDDK (1:1000 dilution, Cat# 14793, RRID:AB_2572291), anti-USP1 (1:1000 dilution, Cat# 8033, RRID:AB_10858879), anti-USP15 (1:1000 dilution, Cat# 66310, RRID:AB_2799706), Rabbit (DA1E) mAb IgG XP® Isotype Control (Cat# 3900, RRID:AB_1550038), Mouse (G3A1) mAb IgG1 Isotype Control (Cat# 5415, RRID:AB_10829607), anti-Lamin B1 (1:1000 dilution, Cat# 13435, RRID:AB_2737428), anti-GAPDH (1:1000 dilution, Cat# 2118, RRID:AB_561053) were purchased from Cell Signaling Technology.

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: Anti-HA-Tag (1:1000 dilution, Cat# 3724, RRID:AB_1549585), anti-GFP-Tag (1:1000 dilution, Cat# 2956, RRID:AB_1196615), anti-T7-Tag (1:1000 dilution, Cat# 13246, RRID:AB_2798161), anti-Myc-Tag (1:1000 dilution, Cat# 2278, RRID:AB_490778), anti-His-Tag (1:1000 dilution, Cat# 12698, RRID:AB_2744546), anti-ubiquitin (1:1000 dilution, Cat# 3936, RRID:AB_331292), anti-PD-L1 (1:1000 dilution, Cat# 13684, RRID:AB_2687655), anti-YTHDF1 (1:1000 dilution, Cat# 86463, RRID:AB_2923055), anti-YTHDF2 (1:1000 dilution, Cat# 80014 RRID:AB_2923056), anti-mTOR (1:1000 dilution, Cat# 2983, RRID:AB_2105622), anti-Phospho-mTOR (Ser2448) (1:1000 dilution, Cat# 5536, RRID:AB_10691552), anti-4E-BP1 (1:1000 dilution, Cat# 9644, RRID:AB_2097841), anti-Phospho-4E-BP1 (Thr37/46) (1:1000 dilution, Cat# 2855, RRID:AB_560835), anti-p70 S6 (1:1000 dilution, Cat# 9202, RRID:AB_331676), anti-Phospho-p70 S6 (Thr389) (1:1000 dilution, Cat# 97596, RRID:AB_2800283), anti-Acetyl-CoA Carboxylase 1 (1:1000 dilution, Cat# 4190, RRID:AB_10547752), anti-Phospho-Acetyl-CoA Carboxylase (Ser79) (1:1000 dilution, Cat# 11818, RRID:AB_2687505), anti-AKT (1:1000 dilution, Cat# 4691, RRID:AB_915783), anti-Phospho-Akt (Ser473) (1:1000 dilution, Cat# 4060, RRID:AB_2315049), anti-SIN (1:1000 dilution, Cat# 12860, RRID:AB_2798048), anti-GβL (1:1000 dilution, Cat# 3274, RRID:AB_823685), anti-Raptor (1:1000 dilution, Cat# 2280, RRID:AB_561245), anti-RICTOR (1:1000 dilution, Cat# 2114, RRID:AB_2179963), anti-DEPTOR (1:1000 dilution, Cat# 11816, RRID:AB_2750575), anti-DYKDDDDK (1:1000 dilution, Cat# 14793, RRID:AB_2572291), anti-USP1 (1:1000 dilution, Cat# 8033, RRID:AB_10858879), anti-USP15 (1:1000 dilution, Cat# 66310, RRID:AB_2799706), Rabbit (DA1E) mAb IgG XP® Isotype Control (Cat# 3900, RRID:AB_1550038), Mouse (G3A1) mAb IgG1 Isotype Control (Cat# 5415, RRID:AB_10829607), anti-Lamin B1 (1:1000 dilution, Cat# 13435, RRID:AB_2737428), anti-GAPDH (1:1000 dilution, Cat# 2118, RRID:AB_561053) were purchased from Cell Signaling Technology.

Techniques: Staining, Expressing, Derivative Assay, Inhibition