cd47  (Cell Signaling Technology Inc)

Journal: Journal for Immunotherapy of Cancer

Article Title: CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy

doi: 10.1136/jitc-2025-013498

Figure Lengend Snippet: USP2 protects CD47 from proteasome-mediated degradation. ( A–C ) IB analysis of WCL collected from MCF7 ( A ), MDA-MB-231 ( B ), or HCT116 cells ( C ) treated with vehicle or the indicated deubiquitinating enzyme inhibitors (10 µM). Indicated proteins were quantified, n=3 biological replicates. ( B ) IB analysis of WCL derived from MDA-MB-231 cells treated with the indicated ML364 concentrations for 24 hours ( h ). Indicated proteins were quantified, n=3 biological replicates. ( E ) CD47 mRNAs derived from MDA-MB-231 cells treated with the indicated ML364 concentrations for 24 hours were determined by RT-qPCR, n=3 biological replicates. ( F ) IB analysis of WCL derived from HCT116 cells treated with the indicated ML364 concentrations for 24 hours. Indicated proteins were quantified, n=3 biological replicates. ( G ) IB analysis of WCL derived from MDA-MB-231 cells treated with 10 µM ML364 for the indicated times. Indicated proteins were quantified, n=3 biological replicates. ( H ) CD47 mRNA expression in MDA-MB-231 cells treated with 10 µM ML364 for the indicated times was determined by RT-qPCR, n=3 biological replicates. ( I ) Flow cytometry analysis of the cell surface CD47 protein expression in MDA-MB-231 cells treated with ML364 (10 µM) for 24 hours. MFI (mean fluorescence intensity) data were quantified. ( J ) IB analysis of WCL derived from HEK293T cells co-transfected with the indicated constructs. WT: wild-type; CA: C276A (enzymatically inactive mutant form of USP2). Indicated proteins were quantified, n=3 biological replicates. ( K ) IB analysis of WCL derived from HEK293T cells co-transfected with the indicated constructs. Cells were treated with the indicated concentrations of ML364 for 24 hours. Indicated proteins were quantified, n=3 biological replicates. ( L ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP or sh USP2 . Indicated proteins were quantified, n=3 biological replicates. ( M ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP or sh USP2 . Cells were treated with 200 µg/mL CHX for the indicated times. ( N ) Quantification of CD47 protein band intensity of IB from MDA-MB-231 cell lysates normalized to vinculin and compared with the t=0 time point, n=3 biological replicates. ( O ) IB analysis of WCL derived from HCT116 cells stably expressing sh GFP or sh USP2 . Cells were treated with 200 µg/mL CHX for the indicated times. ( P ) Quantification of CD47 protein band intensity of IB from HCT116 cell lysates was normalized to vinculin and compared with the t=0 time point, n=3 biological replicates. Data are presented as mean±SD, unpaired two-tailed Student’s t-test. P value<0.05 was considered statistically significant. CHX, cycloheximide; IB, immunoblotting; mRNA, messenger RNA; RT-qPCR, reverse transcription quantitative PCR; USP2, ubiquitin-specific protease 2; WCL, whole-cell lysates; WT, wild-type.

Article Snippet: Anti-CD47 (D3O7P) rabbit mAb (63000, 1:1000), PD-L1 Rabbit antibody (13684, 1:3000), p53 Rabbit antibody (2625, 1:3000), Caspase-3 rabbit mAb (9662, 1:1000), Cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (9664, 1:1000), PARP rabbit mAb (9542, 1:1000), Cleaved PARP rabbit mAb (9541, 1:1000) and GST rabbit antibody (2625, 1:3000) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Transfection, Construct, Mutagenesis, Stable Transfection, Two Tailed Test, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy

doi: 10.1136/jitc-2025-013498

Figure Lengend Snippet: USP2 is a physiological upstream deubiquitinating enzyme for CD47. ( A ) IB analysis of WCL and anti-HA IPs derived from HEK293T cells co-transfected with the indicated constructs. Cells were treated with 10 µM MG132 for 12 hours before harvesting. ( B ) Schematic representation of WT, N-terminal region of the aa 1–266, and C-terminal USP domain of aa 267–605. ( C ) IB analysis of WCL and anti-HA IPs derived from HEK293T cells co-transfected with the indicated constructs. ( D ) IB analysis of WCL and Ni-NTA pull-down products derived from HEK293T cells co-transfected with the indicated constructs and Flag-USP2 (1 µg or 3 µg). Cells were treated with 10 µM MG132 for 12 hours before harvesting. Indicated proteins were quantified, n=3 biological replicates. ( E ) IB analysis of the WCL and Ni-NTA pull-down products derived from HEK293T cells co-transfected with the indicated constructs. Cells were treated with 10 µM ML364 for 12 hours and 10 µM MG132 for 12 hours before harvesting. Indicated proteins were quantified, n=3 biological replicates. ( F ) IB analysis of WCL and Ni-NTA pull-down products derived from HEK293T cells co-transfected with the indicated constructs and Flag-USP2 (1 µg or 3 µg). Cells were treated with 10 µM MG132 for 12 hours before harvesting. Indicated proteins were quantified, n=3 biological replicates. ( G ) IB analysis of WCL derived from HEK293T cells co-transfected with the indicated constructs. Cells were treated with 200 µg/mL CHX for the indicated times. ( H ) Quantification of CD47 protein band intensity from ( G ) normalized to vinculin and compared with the t=0 time point, n=3 biological replicates. ( I ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP , sh USP2 , or sh USP2 +USP2. Indicated proteins were quantified, n=3 biological replicates. ( J ) CD47 mRNAs derived from MDA-MB-231 cells stably expressing shGFP, sh USP2 or sh USP2 +USP2 were determined by RT-qPCR. Indicated proteins were quantified, n=3 biological replicates. ( K ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP or sh USP2 . Cells were treated with 10 µM ML364 for 24 hours before harvesting. Indicated proteins were quantified, n=3 biological replicates. ( L ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP or sh USP2 . Cells were treated with 0.5% DMSO, 10 µM ML364 and 50 µM CQ for 24 hours before harvesting. Indicated proteins were quantified, n=3 biological replicates. Data are presented as the mean±SD, unpaired two-tailed Student’s t-test. P value<0.05 was considered statistically significant. aa, amino acid; CHX, cycloheximide; CQ, chloroquine; DMSO, dimethylsulfoxide; IB, immunoblotting; IP, immunoprecipitates; mRNA, messenger RNA; Ni-NTA, nickel-nitrilotriacetic acid; RT-qPCR, reverse transcription quantitative PCR; USP2, ubiquitin-specific protease 2; WCL, whole-cell lysates; WT, wild-type.

Article Snippet: Anti-CD47 (D3O7P) rabbit mAb (63000, 1:1000), PD-L1 Rabbit antibody (13684, 1:3000), p53 Rabbit antibody (2625, 1:3000), Caspase-3 rabbit mAb (9662, 1:1000), Cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (9664, 1:1000), PARP rabbit mAb (9542, 1:1000), Cleaved PARP rabbit mAb (9541, 1:1000) and GST rabbit antibody (2625, 1:3000) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Transfection, Construct, Stable Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy

doi: 10.1136/jitc-2025-013498

Figure Lengend Snippet: SPOP mediates USP2 proteasomal destruction and subsequently influences CD47 protein abundance. ( A ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP or sh SPOP . The indicated proteins were quantified, n=3 biological replicates. ( B ) USP2 mRNA derived from MDA-MB-231 cells stably expressing the indicated plasmids was determined by RT-qPCR, n=3 biological replicates. ( C ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing the indicated plasmids. Indicated proteins were quantified, n=3 biological replicates. ( D ) USP2 mRNA derived from MDA-MB-231 cells stably expressing the indicated plasmids was determined by RT-qPCR, n=3 biological replicates. ( E ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing shGFP or shSPOP. Cells were treated with 200 µg/mL CHX for the indicated times. ( F ) Quantification of USP2 protein band intensity from MDA-MB-231 cells was normalized to vinculin and compared with the t=0 time point, n=3 biological replicates. ( G ) IB analysis of WCL derived from HCT116 cells stably expressing sh GFP or sh SPOP . Cells were treated with 200 µg/mL CHX for the indicated times. ( H ) Quantification of USP2 protein band intensity from HCT116 cells, normalized to vinculin and compared with the t=0 time point, n=3 biological replicates. ( I ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing sh GFP , sh USP2, and sh USP2 +sh SPOP . Indicated proteins were quantified, n=3 biological replicates. ( J ) IB analysis of WCL derived from MDA-MB-231 cells stably expressing shGFP, shSPOP, and shSPOP+shUSP2. Indicated proteins were quantified, n=3 biological replicates. ( K ) A working model of how CD47 protein stability is regulated by the SPOP-USP2 axis. Data are presented as the mean±SD, unpaired two-tailed Student’s t-test. P value<0.05 was considered statistically significant. CHX, cycloheximide; IB, immunoblotting; mRNA, messenger RNA; RT-qPCR, reverse transcription quantitative PCR; SPOP, speckle-type POZ protein; USP2, ubiquitin-specific protease 2; WCL, whole-cell lysates.

Article Snippet: Anti-CD47 (D3O7P) rabbit mAb (63000, 1:1000), PD-L1 Rabbit antibody (13684, 1:3000), p53 Rabbit antibody (2625, 1:3000), Caspase-3 rabbit mAb (9662, 1:1000), Cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (9664, 1:1000), PARP rabbit mAb (9542, 1:1000), Cleaved PARP rabbit mAb (9541, 1:1000) and GST rabbit antibody (2625, 1:3000) were purchased from Cell Signaling Technology.

Techniques: Quantitative Proteomics, Derivative Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy

doi: 10.1136/jitc-2025-013498

Figure Lengend Snippet: USP2 inhibition promotes antitumor effects that are mainly dependent on CD47 protein expression to impact macrophage function. ( A ) Schematic treatment plan for immunocompetent BALB/cJ mice bearing CT26 ( Cd47 WT or KO) tumors. Mice were subcutaneously implanted with 1×10 5 CT26 cells and treated with vehicle+rat IgG2a clone 2A3 (200 µg per mouse), USP2 inhibitor (ML364, 10 mg/kg), anti-PD-1 mAb clone 29F.1A12 (200 µg per mouse), or combined treatment, as indicated. ( B ) Mice bearing CT26-implanted tumors were assigned to different treatment groups, as indicated. Tumor volumes were measured every 3 days and plotted individually. ( C ) Kaplan-Meier survival curves for mice bearing CT26-implanted tumors in each group treated in ( A ). Gehan-Breslow-Wilcoxon test, p<0.05 was considered statistically significant. ( D ) Schematic treatment plan for immunocompetent BALB/cJ mice bearing CT26. Mice were subcutaneously implanted with 1×10 5 CT26 cells and treated with anti-F4/80-mAb (100 µg per mouse), vehicle+rat IgG2a clone 2A3 (200 µg per mouse), USP2 inhibitor (ML364, 10 mg/kg), anti-PD-1 mAb clone 29F.1A12 (200 µg per mouse), or combined treatment, as indicated. ( E ) Mice bearing CT26-implanted tumors were assigned to different treatment groups, as indicated. Tumor volumes were measured every 3 days and plotted individually. ( F ) Kaplan-Meier survival curves for mice bearing CT26-implanted tumors in each group treated in ( D ). Gehan-Breslow-Wilcoxon test, p<0.05 was considered statistically significant. ( G ) A proposed model suggests that SPOP/USP2/CD47 axis regulates tumors to anti-PD-1 therapy. Low SPOP or high USP2 expression upregulates CD47 protein levels, thereby suppressing macrophage-mediated phagocytosis of tumor cells, reducing M1 macrophage, dendritic cells, CD8+T cells and increasing M2 macrophage, which promotes resistance to anti-PD-1 immunotherapy. Conversely, USP2 inhibitor (ML364 or MS102) reduces CD47 protein levels, thereby enhancing macrophage phagocytosis of tumor cells, increasing M1 macrophage, dendritic cells, CD8+T cells and reducing M2 macrophage, which promotes responsiveness to anti-PD-1 immunotherapy. Figure created with BioRender.com. KO, knockout; mAb, monoclonal antibody; PD-1, programmed cell death protein 1; SPOP, speckle-type POZ protein; USP2, ubiquitin-specific protease 2; WT, wild-type.

Article Snippet: Anti-CD47 (D3O7P) rabbit mAb (63000, 1:1000), PD-L1 Rabbit antibody (13684, 1:3000), p53 Rabbit antibody (2625, 1:3000), Caspase-3 rabbit mAb (9662, 1:1000), Cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (9664, 1:1000), PARP rabbit mAb (9542, 1:1000), Cleaved PARP rabbit mAb (9541, 1:1000) and GST rabbit antibody (2625, 1:3000) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing, Knock-Out, Ubiquitin Proteomics