cd47 antibody b6h12  (Bio X Cell)

Journal: Advanced Science

Article Title: SMYD3 Promotes Immune Evasion in Clear Cell Renal Cell Carcinoma via SREBP1‐Mediated Transactivation of CD47

doi: 10.1002/advs.202404200

Figure Lengend Snippet: Upregulated CD47 expression is the inhibitory checkpoint target of SMYD3 and is associated with poor overall survival in patients with ccRCC: A) Schematic illustration of immune effector gene analysis affected by SMYD3 in ccRCC cells. B) Heatmap of the expression of these four immune inhibitory checkpoints upon SMYD3 knockdown according to the mRNA sequencing data. C) Changes in the mRNA expression levels of CD47 and CD276 upon SMYD3 knockdown in TK10 and 786‐O cells, as determined by qPCR ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t ‐test. D) CD47 expression on the cell surface was determined by flow cytometry in ccRCC cells stably expressing SMYD3 shRNA or overexpressing SMYD3. E) CD47 transcript expression in multiple renal cancer studies from the GEO database. *** p < 0.001, Student's t ‐test. F) CD47 protein expression in ccRCC patients from the CPTAC database. *** p < 0.001, Student's t ‐test. G) mRNA expression of CD47 in renal tumor tissues and paired normal tissues ( n = 30 pairs). Student's t ‐test. H) Representative IHC staining images of CD47 expression at the protein level in tumors compared with normal tissues from the TMA cohort. I) Kaplan–Meier survival analysis of overall survival according to CD47 mRNA levels in patients with ccRCC from the KM‐plotter database. J) Kaplan–Meier plots comparing the OS of ccRCC patients according to CD47 protein abundance in the TMA cohort. Log‐rank test. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IHC recognized the following targets: SMYD3 (ab187149, Abcam), SREBP1 (14088‐1‐AP, Proteintech), CD47 (NBP2‐31106, Novus), and IL‐4 (66142‐1‐Ig, Proteintech).

Techniques: Expressing, Knockdown, Sequencing, Flow Cytometry, Stable Transfection, shRNA, Immunohistochemistry, Quantitative Proteomics

Journal: Advanced Science

Article Title: SMYD3 Promotes Immune Evasion in Clear Cell Renal Cell Carcinoma via SREBP1‐Mediated Transactivation of CD47

doi: 10.1002/advs.202404200

Figure Lengend Snippet: CD47 promotes Th2 cell infiltration in the TME of ccRCC: A) Th2 cell infiltration analysis on the basis of CD47 mRNA levels by ssGSEA in ccRCC cohorts (TCGA, E‐MATAB‐1980, ICGC‐RECA‐EU, GSE167093 , GSE73731 , and GSE40435 ). Student's t ‐test. B) Representative multiplex immunofluorescence images of ccRCC samples (details given in Table ) displaying 2 TMA cores (Patient 3(high expression of CD47) and Patient 4 (low expression of CD47)) after multispectral imaging and enlarged subsections of the core showing each of the individual markers in the composite image after spectral unmixing. The markers used were as follows: CD4 (Opal 520, pseudocolored green), cytokeratin (Opal 480, pseudocolored cyan), Gata3 (Opal 620, pseudocolored orange), CD47 (Opal 690, pseudocolored red), and DAPI as a nuclear marker (pseudocolored blue). Scale bars: 100 µm. C) Quantification of Th2 cell infiltration in the TMA per tumor area (mm 2 ) in patients with ccRCC ( n = 45). Student's t ‐test. D) Representative bioluminescence images of tumors in BALB/c mice orthotopically engrafted with control or CD47‐knockdown RENCA tumors. E) Burden of control and CD47‐knockdown RENCA tumors as measured by bioluminescence ( n = 8). * p < 0.05, Student's t ‐test. F) Flow cytometry gating and frequency of Th2 cells among total CD4 + T cells in RENCA tumors with stable expression of CD47 shRNA or control shRNA. G) Frequencies of Th2 cells among total CD4 + T cells in RENCA tumors with stable expression of CD47 shRNA or control shRNA ( n = 5). *** p < 0.001, Student's t ‐test. H) Representative bioluminescence images of tumors in BALB/c mice orthotopically engrafted with control, Smyd3‐knockdown, and rescued Cd47‐overexpressing RENCA tumors. I) The burden of control, Smyd3‐knockdown, and rescued Cd47‐overexpression RENCA tumors as measured by bioluminescence ( n = 10). n.s., non‐significant, ** p < 0.01, one‐way ANOVA test. J) Representative images of orthotopic syngeneic mouse tumors stained for IL‐4 and quantification of IHC staining for IL‐4 ( n = 3). n.s., non‐significant, *** p < 0.001, one‐way ANOVA test. K) Frequencies of Th2 cells among total CD4+ T cells in RENCA tumors in indicated groups ( n = 5). n.s., non‐significant, ** p < 0.01, *** p < 0.001, one‐way ANOVA test. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IHC recognized the following targets: SMYD3 (ab187149, Abcam), SREBP1 (14088‐1‐AP, Proteintech), CD47 (NBP2‐31106, Novus), and IL‐4 (66142‐1‐Ig, Proteintech).

Techniques: Multiplex Assay, Immunofluorescence, Expressing, Imaging, Marker, Control, Knockdown, Flow Cytometry, shRNA, Over Expression, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: SMYD3 Promotes Immune Evasion in Clear Cell Renal Cell Carcinoma via SREBP1‐Mediated Transactivation of CD47

doi: 10.1002/advs.202404200

Figure Lengend Snippet: SREBP1 acts as the pivotal downstream transcription factor of SMYD3 to promote immune evasion in ccRCC: A) Schematic illustration of the effects of SMYD3 on transcription factors in ccRCC cells. B) Dysregulation of these two TFs (SREBP1 and MLXIPL) in 3 histological subtypes of RCC from TCGA. KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; KICH: kidney chromophobe. C) SREBP1 transcript expression in multiple renal cancer studies from the GEO database. * p < 0.05, *** p < 0.001, Student's t ‐test. D) SREBP1 mRNA expression in renal tumor tissues and paired normal tissues ( n = 30 pairs). Student's t ‐test. E) Representative IHC images of SREBP1 expression at the protein level in tumors compared with normal tissues from the TMA cohort. F) Changes in the mRNA expression level of CD47 upon SREBP1 knockdown in TK10 and 786‐O cells, as determined by qPCR ( n = 3). Data are presented as mean ± SEM of three experiments. ** p < 0.01, *** p < 0.001, Student's t ‐test. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IHC recognized the following targets: SMYD3 (ab187149, Abcam), SREBP1 (14088‐1‐AP, Proteintech), CD47 (NBP2‐31106, Novus), and IL‐4 (66142‐1‐Ig, Proteintech).

Techniques: Expressing, Knockdown

Journal: Advanced Science

Article Title: SMYD3 Promotes Immune Evasion in Clear Cell Renal Cell Carcinoma via SREBP1‐Mediated Transactivation of CD47

doi: 10.1002/advs.202404200

Figure Lengend Snippet: SMYD3 epigenetically activates the transcription of SREBP1, and CD47 is a transcriptional target of SREBP1 in ccRCC cells: A) Changes in SREBP1 mRNA expression levels upon SMYD3 knockdown in TK10 and 786‐O cells ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t test. B) Changes in SREBP1 and CD47 mRNA expression levels upon overexpressiong SMYD3 or methyltransferase inactive mutant of SMYD3 (mSMYD3) in TK10 and 786‐O cells. as determined by qPCR ( n = 3). ** p < 0.01, *** p < 0.001, one‐way ANOVA test. C) SREBP1 and CD47 protein expression level changes upon SMYD3 knockdown and overexpression in TK10 and 786‐O cells. D) SREBP1 promoter luciferase activity analysis upon SMYD3 knockdown and overexpression in TK10 and 786‐O cells ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test. E) Luciferase activity analysis of wild‐type (WT) or SMYD3 motif mutants (MT1–MT5) regulating the SREBP1 promoter in ccRCC cells ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, Student's t test. F) Quantitative ChIP assay for H3‐K4 tri‐/di‐/monomethylation and Sp1 and SMYD3 occupancy at site 4 of the SREBP1 promoter in ccRCC cells expressing sh‐SMYD3 or control shRNA ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t‐ test. G) Quantitative ChIP assay for H3‐K4 tri‐/di‐/monomethylation and Sp1 and SMYD3 occupancy at site 5 of the SREBP1 promoter in ccRCC cells expressing sh‐SMYD3 or control shRNA ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t test. H) Quantitative ChIP assay for H3‐K4 tri‐/di‐/monomethylation and Sp1 and SMYD3 occupancy at site 4 of the SREBP1 promoter in ccRCC cells upon expression of SMYD3, mSMYD3 or control ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test. I) Quantitative ChIP assay for H3‐K4 tri‐/di‐/monomethylation and Sp1 and SMYD3 occupancy at site 5 of the SREBP1 promoter in ccRCC cells upon expression of SMYD3, mSMYD3 or control ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test. J) CD47 protein expression changes upon SREBP1 knockdown and overexpression in TK10 and 786‐O cells. K) CD47 expression on the cell surface of ccRCC cells stably expressing SREBP1 shRNA, cDNA, or control was determined by flow cytometry. L) CD47 promoter luciferase activity analysis upon SREBP1 knockdown and overexpression in TK10 and 786‐O cells ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t ‐test. M) Luciferase activity analysis of wild‐type (WT) or SREBP1 motif mutants (MT1 and MT2) regulating the CD47 promoter in ccRCC cells ( n = 3). ** p < 0.01, *** p < 0.001, Student's t ‐test. N) Quantitative ChIP assay for SREBP1 occupancy at the SREs of CD47 promoter in RCC cells ( n = 3). ** p < 0.01, *** p < 0.001, Student's t ‐test. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IHC recognized the following targets: SMYD3 (ab187149, Abcam), SREBP1 (14088‐1‐AP, Proteintech), CD47 (NBP2‐31106, Novus), and IL‐4 (66142‐1‐Ig, Proteintech).

Techniques: Expressing, Knockdown, Mutagenesis, Over Expression, Luciferase, Activity Assay, Control, shRNA, Stable Transfection, Flow Cytometry

Journal: Advanced Science

Article Title: SMYD3 Promotes Immune Evasion in Clear Cell Renal Cell Carcinoma via SREBP1‐Mediated Transactivation of CD47

doi: 10.1002/advs.202404200

Figure Lengend Snippet: SMYD3 protects renal cancer cells from phagocytosis by monocyte‐derived macrophages, and pharmacological inhibition of SMYD3 enhances the anti‐PD‐1 response: A) The indicated TK10 and 786‐O cells were labeled with the fluorescent dye CFSE, incubated with human peripheral blood monocyte‐derived macrophages for 2 h, and stained with CD11b and analyzed by flow cytometry. The phagocytosis rate was calculated as the percentage of CFSE + CD11b + cells in CSFE + cells ( n = 3), and the phagocytosis rate in control cells was set as 100%. ** p < 0.01, Student's t ‐test. B) The inhibitory effect on CD47 expression caused by SMYD3 knockdown could be reversed by SREBP1 overexpression in ccRCC cells. C) The decrease in CD47 expression on the cell surface caused by SMYD3 knockdown was abolished by SREBP1 overexpression in ccRCC cells. D) The indicated TK10 and 786‐O cells were subjected to a phagocytosis assay similar to that described in A ( n = 3). n.s., non‐significant, * p < 0.05, ** p < 0.01, one‐way ANOVA test. E) Representative bioluminescence images of tumors in BALB/c mice orthotopically engrafted with control, SMYD3‐knockdown, and rescued SREBP1‐overexpressing RENCA tumors. F) Burden of control, SMYD3‐knockdown, and rescued SREBP1‐overexpressing RENCA tumors as measured by bioluminescence ( n = 10). n.s., non‐significant, * p < 0.05, one‐way ANOVA test. G) Flow cytometry gating and frequency of Th2 cells among total CD4 + T cells in RENCA tumors in the indicated groups ( n = 5). n.s., non‐significant, ** p < 0.01, one‐way ANOVA test. H) Representative bioluminescence image of Day 16 tumors in mice with RENCA tumors treated with vehicle, the SMYD3 inhibitor BCI‐121 (1 mg kg −1 , i.p., daily), anti‐PD‐1 mAb (10 mg kg −1 , i.p., on Days 1, 4, 7 and 14) or a combination of BCI‐121 and anti‐PD‐1 mAb. I) Burden of RENCA tumors treated with vehicle, BCI‐121, an anti‐PD‐1 mAb, or a combination of both ( n = 10). n.s., non‐significant, * p < 0.05, ** p < 0.01, one‐way ANOVA test. J) Flow cytometry gating and frequency of Th2 cells among total CD4 + T cells in RENCA tumors treated with either vehicle, BCI‐121, anti‐PD‐1 mAb, or a combination of BCI‐121 and anti‐PD‐1 mAb ( n = 5). n.s., non‐significant, *** p < 0.001, one‐way ANOVA test. K) Representative images of orthotopic syngeneic mouse tumors stained for SMYD3, SREBP1, CD47, and IL‐4 and quantification of IHC staining ( n = 3). n.s., non‐significant, ** p < 0.01, *** p < 0.001, one‐way ANOVA test. L) Proposed schematic representation depicting the mechanism by which SMYD3 transcriptionally activates SREBP1‐CD47 to promote Th2 cell infiltration‐induced immune evasion in ccRCC. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IHC recognized the following targets: SMYD3 (ab187149, Abcam), SREBP1 (14088‐1‐AP, Proteintech), CD47 (NBP2‐31106, Novus), and IL‐4 (66142‐1‐Ig, Proteintech).

Techniques: Derivative Assay, Inhibition, Labeling, Incubation, Staining, Flow Cytometry, Control, Expressing, Knockdown, Over Expression, Phagocytosis Assay, Immunohistochemistry