Journal: Theranostics

Article Title: USP7 targeting modulates anti-tumor immune response by reprogramming Tumor-associated Macrophages in Lung Cancer

doi: 10.7150/thno.47137

Figure Lengend Snippet: Targeting USP7 inhibits murine M2 phenotype and function in vitro . (A) The mRNA expression of common genes related to DUBs was detected by RT-PCR in M0, M1 (LPS/IFN-γ M1), and M2 (IL-4/13 M2 and IL-10 M2) induced from ANA-1. ( B ) The mRNA expression of USP7 in MΦs, M1, and M2 induced from BMDMs was detected by RT-PCR. ( C ) Western blotting showing the expression of USP7 in MΦ, M1, and M2 induced from BMDMs. ( D-E ) Flow cytometry analyses of the expression of CD206 in IL-4/13-induced BMDM M2 cells, which had been treated with P5091 (5 μM or 10 μM) for 24 h. Data are presented as the mean ± SEM (n = 3). ( F ) Detection of the expression of USP7 in BMDMs, which were transfected with either NC- or USP7-siRNA by western blotting. ( G ) The expression of CD206 in BMDMs of NC-siRNA and USP7-siRNA group was detected by flow cytometry. ( H-I ) Flow cytometry analyses of CFSE expression on the surface of CD8 + T cells in the presence of conditioned medium from either DMSO- or P5091- (5 μM or 10 μM) treated IL-4/13-induced BMDM M2 cells. Data are presented as the mean ± SEM (n = 3).

Article Snippet: CD8 antibody was purchased from BOSTER Biological Technology Co., Ltd (Wuhan, China).

Techniques: In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Transfection

Journal: Theranostics

Article Title: USP7 targeting modulates anti-tumor immune response by reprogramming Tumor-associated Macrophages in Lung Cancer

doi: 10.7150/thno.47137

Figure Lengend Snippet: Targeting USP7 inhibits tumor growth and induces local anti-tumor immunity in vivo . ( A ) Harvested and photographed tumors in the P5091 and the Control group. ( B ) Lewis tumor growth following P5091 or Vehicle treatment in vivo. Data are presented as the mean ± SEM (n = 6 per group). ( C ) Spider diagram of the tumor volume growth in each mouse from the P5091 group and the Control group. ( D ) Gating strategy for detection of the TAMs by flow cytometry. ( E-G ) Proportions of M1 ( E ) and M2 ( F ), and M1/M2 ratio ( G ) in the TME of the P5091 group and the Control group. ( H-M ) Percentages of MDSC ( H ), Treg ( I ), CD4 + T ( J ), CD8 + T ( K ), Th1 ( L ), and CTLs ( M ) within the TME in each group. Data are presented as the mean ± SEM (n = 7) for ( E-M ).

Article Snippet: CD8 antibody was purchased from BOSTER Biological Technology Co., Ltd (Wuhan, China).

Techniques: In Vivo, Flow Cytometry

Journal: Theranostics

Article Title: USP7 targeting modulates anti-tumor immune response by reprogramming Tumor-associated Macrophages in Lung Cancer

doi: 10.7150/thno.47137

Figure Lengend Snippet: Targeting USP7 activates local and systemic anti-tumor immunity. ( A ) Multicolor immunofluorescence detection of TAMs and CTLs in the TME of the P5091 group and the Control group. ( B-F ) Cytokines IFN-γ ( B ), TNF-α ( C ), IL-2 ( D ), IL-5 ( E ), and IL-6 ( F ) in the TME of each group were detected by Mul-Analyte Flow Assay Kit. ( G-K ) Proportion of Treg ( G ), CD4 + T ( H ), CD8 + T ( I ), Th1 ( J ), and CTLs ( K ) in the spleen of each group. Data are presented as the mean ± SEM (n = 7) for ( B-K ).

Article Snippet: CD8 antibody was purchased from BOSTER Biological Technology Co., Ltd (Wuhan, China).

Techniques: Immunofluorescence

Journal: Theranostics

Article Title: USP7 targeting modulates anti-tumor immune response by reprogramming Tumor-associated Macrophages in Lung Cancer

doi: 10.7150/thno.47137

Figure Lengend Snippet: Targeting USP7 can activate the p38 MAPK pathway to reprogram TAMs. ( A ) The strategy for sorting TAMs in TME by flow cytometry. ( B ) Heat maps illustrating the differentially expressed M1- and M2-related genes in TAMs between the P5091 group and the Control group based on the results of RNA sequencing. ( C ) RT-PCR further verifying the differentially expressed genes of sorted TAMs in each group. Data are presented as the mean ± SEM (n = 3). ( D ) KEGG analysis identifying 20 most obviously enriched pathways based on the differentially expressed genes of the two groups. ( E ) Western blotting detection of the expression of JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, and β-actin in IL-4/13-BMDM M2 cells treated with P5091 (10 µM) at the indicated time points. ( F ) Flow cytometry analysis of CFSE expression on the surface of CD8 + T cells in the presence of conditioned medium of IL-4/13-induced BMDM M2 cells from various indicated treatments. Treatments indicated: DMSO stimulation, P5091 (10 µM) stimulation, P5091 (10 µM) stimulation in the presence of inhibitors of p38 (SB203580, 10 µM), JNK (SP600125, 10 µM), Erk1/2 (U0126-EtOH, 10 µM). Data are presented as the mean ± SEM (n = 4).

Article Snippet: CD8 antibody was purchased from BOSTER Biological Technology Co., Ltd (Wuhan, China).

Techniques: Flow Cytometry, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing