Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 2.5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) five times at three-day intervals. Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05 and ** p < 0.01. Two-Way ANOVA test ( n = 4). B The resected tumors were weighed on Day 30. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. D The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). E The mRNA levels of Cd86 and Cd163 in resected tumors were analyzed by qRT‒PCR ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). F The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry ( n = 3-4). * p < 0.05 and *** p < 0.001. One-Way ANOVA test. G The quantification of the M1/M2 ratio is shown ( n = 3). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). H The frequencies of CD4 (CD4 + CD3 + CD45 + 7AAD - ) and CD8 (CD8 + CD3 + CD45 + 7AAD - ) tumor-infiltrating T cells were analyzed by flow cytometry ( n = 3-4). * p < 0.05. One-Way ANOVA test. I The quantification of CD8 + TILs is shown. * p < 0.05. One-Way ANOVA test ( n = 3). J The frequency of regulatory CD4 T cells (CD25 + CD127 - CD4 + CD3 + CD45 + 7AAD - ) was analyzed by flow cytometry ( n = 3-4). K The quantification of the CD8/Treg ratio is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). L The densities of dendritic cells (CD11c + ) and cytotoxic T cells (CD8 + ) in resected tumors were analyzed by immunofluorescence staining. The density of CD11c + DCs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). M The density of CD8 + TILs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3).

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Immunofluorescence, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 5 days and then intraperitoneally administered with anti-ENO1 antibodies (HuL001, 40 mg/kg) or clodronate liposomes (50 μL/mouse) on the indicated days ( n = 5). Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 4). B The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C 4T1 cells (5 ×10 4 ) were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) six times on the indicated days ( n = 5). Local radiotherapy was given on Days 10 and 12. The tumor volume was recorded every three days. * p < 0.05 and *** p < 0.001. Two-Way ANOVA test ( n = 4). D The resected tumors were weighed on Day 40. * p < 0.05. One-Way ANOVA test ( n = 4). E The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. F The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05. One-Way ANOVA test ( n = 3). G The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD1206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). H The quantification of the M1/M2 ratio is shown. * p < 0.05. One-Way ANOVA test ( n = 3). I A representative image of flow cytometric analysis of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells. J The frequency of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells was analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). K The density of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown. * p < 0.05. One-Way ANOVA test ( n = 3-4). L The density of IFNγ + (IFNγ hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown ( n = 3-4). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3-4). M The proposed mechanism of TGFβ1/TGFβR/Smad3/PRMT5-mediated ENO1 translocation for lactate release via MCT4.

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Liposomes, Immunofluorescence, Staining, Flow Cytometry, Translocation Assay

Journal: Journal of Biological Chemistry

Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to N-Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E

doi: 10.1074/jbc.m111.243410

Figure Lengend Snippet: FIGURE 6. Siglec-E-Fc binds subsets of sialylated proteins from activated T-lymphocytes. A, purified T cells were activated for 24 h and either untreated or sialidase-treated and then surface-labeled with biotin. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc com- plexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with streptavidin-HRP to detect surface biotinylated proteins. Arrows indicated proteins that bound to Siglec-E-Fc in a sialidase-sensitive manner (lane 4). B, purified T cells were activated for 24 h and treated or not with sialidase and lysates passed over MAL or SNA affinity columns. Bound sialylated proteins were eluted with SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with siglec-E-Fc precomplexed to anti-Fc-HRP. Arrows indicate proteins that are recognized by siglec-E-Fc in a sialidase-sensitive manner. C, purified T cells were activated for 24 h, cultured for 4 days and treated or not with sialidase. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc complexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with goat anti-CD45 followed by HRP-conjugated anti-goat secondary antibody. The arrow indicates CD45 that is recognized by siglec-E-Fc in a sialidase-sensitive manner. The other bands represent nonspecifically bound proteins.

Article Snippet: For CD45 blots, membranes were blocked as above and then probed with 1 g/ml goat anti-mouse CD45 primary antibody and 1:10000 HRP-conjugated anti-goat IgG secondary antibody (R&D Systems) and detected as described previously.

Techniques: Purification, Labeling, SDS Page, Western Blot, Cell Culture