Journal: The FASEB Journal

Article Title: Nur77 Regulates the Phosphorylation of Smad3, Thereby Influencing Skeletal Muscle Fibrosis Caused by Obesity

doi: 10.1096/fj.202502392RR

Figure Lengend Snippet: Nur77 binds to Smad3 to regulate indicators of skeletal muscle fibrosis. (A, B) Western blot was used to detect the levels of total Smad3 and p‐Smad3 in the cases of Nur77 overexpression and knockout; (C, D) Co‐IP experiments were conducted to examine the binding of Nur77 to Smad3; (E) Cell immunofluorescence was employed to detect the co‐localization of Smad3 and Nur77. (F) Flag‐Smad3 fusion proteins were incubated with bacterially expressed GST‐Nur77 fragments in vitro for GST‐pull down assay. CBB staining indicates the expression of GST and GST‐Nur77 fragments. (G) In the case of overexpression of Nur77, the proliferation of satellite cells was detected by Brdu staining. (H–K) Western blot was used to detect the expression levels and quantities of collagen、Nur77 and Smad3 and Smad3 under TGF‐β stimulation at different times; (L–O) Western blot detected the expression of smad3 and its phosphorylation, as well as the expression of collagen under the influence of sis3. (P) Western blot detected the levels of AKT, IR, GSK‐3β and their phosphorylation under insulin stimulation. (Q–T) Western blot was used to detect the expression levels and quantities of collagen and Nur77 under the action of different concentrations of Csn‐B under TGF‐β stimulation. The data were expressed as the mean ± standard deviation (SD) ( n = 3), with **** indicating p < 0.0001, *** indicating p < 0.001, ** indicating p < 0.01, and * indicating p < 0.05.

Article Snippet: Rabbit anti‐Collagen I a1 (1:1000, Zenbio, R26615 ), Rabbit anti‐Collagen III a1 (1:1000, Proteintech, 22 734‐1‐AP), Rabbit anti‐Smad3 (1:1000, Cell Signaling Technology, 9523), Rabbit anti‐p‐Smad3 (1:1000, Cell Signaling Technology, 9520), Rabbit anti‐TGF‐β1 (1:1000, Abcam, ab142139), Rabbit anti‐Nur77 (Novusbio, JM59‐11), Rabbit anti‐pax7 (1:1000, Abcam ab211542), Rabbit anti‐myod (1:1000, SantaCrue sc‐32 758), Rabbit anti‐myog (1:1000, SantaCrue sc‐52 903), Rabbit anti‐GAPDH (1:1000, Cell Signaling Technology, 92 310), Rabbit anti‐AKT (1:1000, Cell Signaling Technology, 9272), Rabbit anti‐Phospho‐AKT (ser473) (1:1000, Cell Signaling Technology, 4060), Rabbit anti‐GSK‐3β (1:1000, Cell Signaling Technology, 12 456), Rabbit anti‐Phospho‐GSK‐3β (1:1000, Cell Signaling Technology,5558),PA (palmitic acid)was purchased from Solarbio (IP0670,Beijing,China), SIS3 was purchased from MCE (HY‐13013).

Techniques: Western Blot, Over Expression, Knock-Out, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Incubation, In Vitro, Pull Down Assay, Staining, Expressing, BrdU Staining, Phospho-proteomics, Standard Deviation

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 The expression of total and surface ENO1 in human CRC cell lines was evaluated by immunoblotting and flow cytometry. B The expression of total and surface ENO1 in human BRCA cell lines was evaluated by immunoblotting and flow cytometry. C HT29 and LoVo cells were treated with different doses of recombinant human TGFβ1 protein (rhTGFβ1) for 24 hr, after which surface ENO1 expression was analyzed via flow cytometry. D HS578T and MDA-MB-468 cells were treated with different doses of the rhTGFβ1 protein for 24 hr, after which the surface ENO1 level was analyzed via flow cytometry. E LoVo cells were treated with LPS (1 μg/mL), rhTGFβ1 protein (10 ng/mL) or H 2 O 2 (50 μM) in combination with a PRMT5 inhibitor (AMG-193, 1 μM) or a PRMT6 inhibitor (EPZ020411 hydrochloride, 1 μM) for 24 hr. The surface ENO1 level was analyzed via flow cytometry. * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). F HT29 cells were treated with rhTGFβ1 protein in combination with a PRMT5 inhibitor (1 μM) for 24 hr. The surface ENO1 expression was analyzed via flow cytometry. ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). G LoVo shNC and LoVo shPRMT5 cells were treated with LPS (1 μg/mL), rhTGFβ1 protein (10 ng/mL) or H 2 O 2 (50 μM) for 24 hr. The surface level of ENO1 was examined by flow cytometry. * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). H HT29 and LoVo cells were treated with LPS (1 μg/mL) and RT (5 Gy) for 24 hr. The surface level of ENO1 was examined by flow cytometry. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). I HT29 cells were treated with RT (5 Gy) in combination with a TGFβR1 inhibitor (1 μM), a Smad3 inhibitor (1 μM), or a PRMT5 inhibitor (1 μM) for 24 hr. The surface level of ENO1 was examined by flow cytometry. *** p < 0.001. One-Way ANOVA test ( n = 3). J HT29 cells were treated with RT (5 Gy) in combination with a TGFβR1 inhibitor (1 μM), a Smad3 inhibitor (1 μM), or a PRMT5 inhibitor (1 μM) for 24 hr. The level of plasmin activity was examined via an ELISA kit. * p < 0.05. One-Way ANOVA test ( n = 3). K MDA-MB-468 cells were treated with RT (5 Gy) in combination with a TGFβR1 inhibitor (1 μM), a Smad3 inhibitor (1 μM), or a PRMT5 inhibitor (1 μM) for 24 hr. The surface level of ENO1 was examined by flow cytometry. * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). L MDA-MB-468 cells were treated with RT (5 Gy) in combination with a TGFβR1 inhibitor (1 μM), a Smad3 inhibitor (1 μM), or a PRMT5 inhibitor (1 μM) for 24 hr. The level of plasmin activity was examined via an ELISA kit. * p < 0.05, and ** p < 0.01. One-Way ANOVA test ( n = 3). M HT29 cells were treated with RT (5 Gy) in combination with a PRMT5 inhibitor (1 μM) for 24 hr. The level of surface-methylated ENO1 was evaluated by immunoprecipitation and immunoblotting.

Article Snippet: The TGFβR1 inhibitor galunisertib (HY-13226, MCE, USA) [ ], Smad3 inhibitor SIS3 (HY-13013, MCE, USA) and PRMT5 inhibitor EPZ015666 (HY-12727, MCE, USA) were dissolved in DMSO to a concentration of 10 mM.

Techniques: Expressing, Western Blot, Flow Cytometry, Recombinant, Activity Assay, Enzyme-linked Immunosorbent Assay, Methylation, Immunoprecipitation

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 TGFβR1 inhibitor galunisertib (HY-13226, MCE, USA) [ ], Smad3 inhibitor SIS3 (HY-13013, MCE, USA) and PRMT5 inhibitor EPZ015666 (HY-12727, MCE, USA) were dissolved in DMSO to a concentration of 10 mM.

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