Journal: Advanced Science

Article Title: EGFR‐Induced and c‐Src‐Mediated CD47 Phosphorylation Inhibits TRIM21‐Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion

doi: 10.1002/advs.202206380

Figure Lengend Snippet: c‐Src activation induces CD47 protein upregulation in response to EGFR activation. Immunoblot analyses were performed with the indicated antibodies (A–E). A) Serum‐starved U251 cells were pretreated with the indicated inhibitors for 2 h and then stimulated with EGF (100 ng mL −1 ) for 24 h. B) Serum‐starved U251 cells with stable expression of different shRNAs against c‐Src or a control shRNA were stimulated with or without EGF (100 ng mL −1 ) for 24 h. C) U251 cells were transfected with a control vector, wild‐type (WT) c‐Src, or an active c‐Src (CA) for 48 h. D) U87/EGFRvIII cells were pretreated with DMSO or Su6656 (4 µ m ) for 2 h and then treated with CHX (100 µg mL −1 ) for the indicated periods of time. Quantification of relative CD47 protein levels is shown (bottom panel). E) U251 cells stably expressed c‐Src WT or c‐Src CA were treated with CHX (100 µg mL −1 ) for the indicated periods of time. Quantification of relative CD47 protein levels is shown (right panel).

Article Snippet: Mouse monoclonal antibodies for FLAG (F3165, M2, 1:5000 for immunoblotting, 1:1000 for immunoprecipitation) and polyclonal antibodies for c‐Src (pY416, SAB5701768, 1:1000 for immunoblotting, 1:100 for IHC) were purchased from Sigma (St. Louis, MO).

Techniques: Activation Assay, Western Blot, Expressing, shRNA, Transfection, Plasmid Preparation, Stable Transfection

Journal: Advanced Science

Article Title: EGFR‐Induced and c‐Src‐Mediated CD47 Phosphorylation Inhibits TRIM21‐Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion

doi: 10.1002/advs.202206380

Figure Lengend Snippet: c‐Src binds to and phosphorylates CD47 at Y288, which subsequently upregulates CD47 stability by inhibiting CD47 polyubiquitylation. Immunoblotting analyses were performed with the indicated antibodies (A–K). A) Serum‐starved U251 cells were stimulated with or without EGF (100 ng mL −1 ) for 1 h. Endogenous CD47 was immunoprecipitated. B) U251 cells were transfected with c‐Src WT or c‐Src CA for 48 h. Endogenous CD47 was immunoprecipitated. C) In vitro kinase assays were performed by mixing purified WT His‐CD47 or His‐CD47 Y288F with or without active c‐Src. D) Serum‐starved U251 cells were stimulated with or without EGF (100 ng mL −1 ) for 1 h. E) Serum‐starved U251 cells were stimulated with or without EGF (100 ng mL −1 ) for 1 h in the presence or absence of the indicated inhibitors. F) HEK293T/EGFR cells were transiently transfected with WT Flag‐CD47 or the indicated Flag‐tagged mutants and stimulated with or without EGF (100 ng mL −1 ) for 24 h. G) HEK293T/EGFR cells were transiently transfected with WT Flag‐CD47 or the indicated Flag‐tagged mutants and stimulated with or without EGF (100 ng mL −1 ) for 24 h (left panel) or transiently co‐transfected with WT Flag‐CD47 or the indicated Flag‐tagged mutants and wild‐type or c‐Src CA for 48 h (right panel). H) CD47 knockdown U251 cells with reconstituted expression of WT Flag‐rCD47 or Flag‐rCD47 Y288F mutant were transfected with HA‐Ub and then stimulated with or without EGF (100 ng mL −1 ) for 60 min. MG132 (10 µ m ) was added to the cells 6 h before they were harvested with guanidine‐HCl‐containing buffer. Immunoprecipitation of Flag was performed with an anti‐Flag antibody. I) HEK293T/EGFR cells were co‐transfected with c‐Src WT or c‐Src CA, CD47‐Flag, and HA‐Ub. The cells were harvested with a guanidine‐HCl‐containing buffer. Immunoprecipitation was performed with an anti‐Flag antibody. J) U87/EGFRvIII cells were co‐transfected with control shRNA, c‐Src shRNA, or HA‐Ub. MG132 (10 µ m ) was added to the cells 6 h before they were harvested with guanidine‐HCl‐containing buffer. Immunoprecipitation was performed with an anti‐CD47 antibody. K) CD47‐depleted U87/EGFRvIII cells with reconstituted expression of WT Flag‐rCD47 or Flag‐rCD47 Y288F mutant were treated with CHX (100 µg mL −1 ) for the indicated periods of time. Quantification of relative Flag (rCD47) protein levels is shown (bottom panel).

Article Snippet: Mouse monoclonal antibodies for FLAG (F3165, M2, 1:5000 for immunoblotting, 1:1000 for immunoprecipitation) and polyclonal antibodies for c‐Src (pY416, SAB5701768, 1:1000 for immunoblotting, 1:100 for IHC) were purchased from Sigma (St. Louis, MO).

Techniques: Western Blot, Immunoprecipitation, Transfection, In Vitro, Purification, Expressing, Mutagenesis, shRNA

Journal: Advanced Science

Article Title: EGFR‐Induced and c‐Src‐Mediated CD47 Phosphorylation Inhibits TRIM21‐Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion

doi: 10.1002/advs.202206380

Figure Lengend Snippet: EGFR activation‐induced and c‐Src‐mediated CD47 phosphorylation and stabilization promote immune evasion of tumor cells and brain tumor growth. A) A total of 1 × 10 5 mouse CT‐2A/Luc‐GFP cells with or without knockout of CD47 or knock‐in of CD47 Y286F or K99/102R mutant were intracranially injected into C57BL/6 mice. After 15 days, the mice were euthanized and examined for tumor growth. Representative tumor growth was shown in vivo by bioluminescence imaging using IVIS 100. B) A bioluminescence imaging analysis of tumor burden was performed on the indicated days. C) The mouse survival times were recorded and visualized using Kaplan–Meier survival curves. D) Immunofluorescent staining of the mouse GBM specimens was performed with the indicated antibodies. The macrophages that engulfed the cancer cells were indicated with arrows. Scale bar, 50 µm. E) Tumor macrophage phagocytosis was estimated by quantification of the phagocytic index ( n = 4). The results represent the means ± SD; ANOVA two‐way test. Different letters indicate significant differences ( p < 0.05). F) A total of 1 × 10 5 CT‐2A/Luc‐GFP cells were intracranially injected into syngeneic C57BL/6 mice. After 15 days, the mice were euthanized and examined for tumor growth. Representative tumor growth was shown in vivo by bioluminescence imaging using IVIS 100. G) A bioluminescence imaging analysis of tumor burden was performed on the indicated days. H) The mouse survival times were recorded and visualized using Kaplan–Meier survival curves. I) Immunofluorescent staining of the mouse GBM specimens was performed with the indicated antibodies. The macrophages that engulfed the cancer cells were indicated with arrows. Scale bar, 50 µm. J) Tumor macrophage phagocytosis was estimated by quantification of the phagocytic index ( n = 4). The results represent the means ± SD; ANOVA two‐way test. Different letters indicate significant differences ( p < 0.05).

Article Snippet: Mouse monoclonal antibodies for FLAG (F3165, M2, 1:5000 for immunoblotting, 1:1000 for immunoprecipitation) and polyclonal antibodies for c‐Src (pY416, SAB5701768, 1:1000 for immunoblotting, 1:100 for IHC) were purchased from Sigma (St. Louis, MO).

Techniques: Activation Assay, Knock-Out, Knock-In, Mutagenesis, Injection, In Vivo, Imaging, Staining

Journal: Advanced Science

Article Title: EGFR‐Induced and c‐Src‐Mediated CD47 Phosphorylation Inhibits TRIM21‐Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Activation of the EGFR/c‐Src pathway correlates with CD47 expression in human GBM specimens. A) IHC staining of 25 human GBM specimens was performed with the indicated antibodies. Representative images from the staining of seven different specimens are shown. Scale bar, 100 µm. B,C) The IHC stains were scored, and correlation analyses were performed. The Pearson correlation test was used. Note that the scores of some samples overlap. D) A schematic of c‐Src‐regulated CD47 phosphorylation and expression.

Article Snippet: Mouse monoclonal antibodies for FLAG (F3165, M2, 1:5000 for immunoblotting, 1:1000 for immunoprecipitation) and polyclonal antibodies for c‐Src (pY416, SAB5701768, 1:1000 for immunoblotting, 1:100 for IHC) were purchased from Sigma (St. Louis, MO).

Techniques: Activation Assay, Expressing, Immunohistochemistry, Staining