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

Journal: Bioactive Materials

Article Title: Hydrogel delivering antifibrotic agent and nano-sonosensitizer enhances efficacy of sonodynamic therapy in osteosarcoma treatment

doi: 10.1016/j.bioactmat.2025.10.001

Figure Lengend Snippet: SIS3 reprograms CAFs (a) Schematic representation depicting the in vitro activation of NIH3T3 cells into CAFs using an OS-conditioned medium. (b) Western blotting is used to assess the expression levels of CAFs activation markers α-SMA and FAP. (c) Immunofluorescence (IF) staining is used to visualize the expression of CAFs activation markers α-SMA and FAP (scale bar: 25 μm). (d) IF staining is used to detect the expression of α-SMA and FAP in NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 25 μm). (e) Statistical analysis of relative fluorescence intensity (FI), presented as mean ± SD (n = 3). (f) Schematic illustration of the TGF-β/SMAD3 signaling pathway. (g) Western blotting is used to evaluate the activation status of the TGF-β/SMAD3 signaling pathway in NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 and the statistical analysis of relative SMAD and p-SMAD expression, presented as mean ± SD (n = 3). (h) Representative images from collagen gel contraction assays on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 and the statistical analysis of relative contraction degree, presented as mean ± SD (n = 3). (i) Representative images from wound healing assays conducted on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 200 μm) and the statistical analysis of relative wound healing degree, presented as mean ± SD (n = 3). (j) Representative images from transwell migration assays performed on NIH3T3 cells, CAFs, and CAFs co-cultured with SIS3 (scale bar: 100 μm) and the statistical analysis of the relative number of migrated cells, presented as mean ± SD (n = 3).

Article Snippet: The primary antibodies used were: α-SMA (1:1000, ABclonal, A17910), FAP (1:1000, ABclonal, A23789 ), SMAD (1:2000, Proteintech, 66516-1-Ig), p-SMAD (S423+S425) (1:2000, Abcam, ab52903), STING (1:1000, Cell Signaling Technology, D2P2F), p-STING (Ser365) (1:1000, Cell Signaling Technology, D8F4W), p-TBK1 (Ser172) (1:1000, Cell Signaling Technology, D52C2), and p-IRF3 (Ser396) (1:1000, Cell Signaling Technology, D6O1M).

Techniques: In Vitro, Activation Assay, Western Blot, Expressing, Immunofluorescence, Staining, Cell Culture, Fluorescence, Migration

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole modulates SMAD3 phosphorylation and nuclear translocation. (A) GES-1 and AGS cells were treated with or without rabeprazole for 1 h, and the phosphorylation of SMAD3 linker was detected by immunoblotting. (B-E) The band intensities were quantified and analyzed by one sample t-test. Data are shown as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001, n=3. (F) Left panel: The subcellular fraction was isolated using nuclear and cytoplasmic protein extraction kit according to manufacturer's instructions. The SMAD3 level was detected by western blotting, α-tubulin and lamin A/C were used as cytosolic and nuclear internal controls. Right panel: IF analysis of SMAD3 in AGS cells treated with or without rabeprazole for 1 h. Scale bar, 100 µm. SMAD3, SMAD family member 3; IF, immunofluorescence; phospho, phosphorylated.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Isolation, Protein Extraction, Immunofluorescence

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole modulates the TIF1γ/SMAD3 complex. (A) Following overnight serum starvation, AGS cells were treated with or without rabeprazole for 1 h, and an IP experiment with anti-SMAD3 or IgG was performed. Immunoblotting was employed to analyze the indicated protein expression levels. (B-E) Band intensities were quantified and the differences were analyzed. Data are shown as the mean ± SD and determined using one sample t-test; * P<0.05 and ** P<0.01; n=3. TIF1γ, transcriptional intermediary factor 1γ; SMAD3, SMAD family member 3; IP, immunoprecipitation.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Western Blot, Expressing, Immunoprecipitation

Journal: World Journal of Stem Cells

Article Title: Extracellular vesicles derived from human amniotic fluid stem cells improve bladder dysfunction in rat model of diabetic atherosclerosis

doi: 10.4252/wjsc.v18.i1.113614

Figure Lengend Snippet: Relative mRNA expressions of tumor necrosis factor-α, interleukin-6, transforming growth factor-β1, Smad3, connective tissue growth factor, and fibronectin in the bladder of control and diabetic groups. A-F: Compared with the control group, diabetes mellitus and diabetic atherosclerosis (DMA) bladders showed significant increases in the mRNA expression of tumor necrosis factor α ( TNF-α ), interleukin-6 ( IL-6 ), transforming growth factor β1 ( TGF-β1 ), Smad3 , connective tissue growth factor ( CTGF ), and fibronectin at 4 weeks and 12 weeks after induction. Compared with the diabetes mellitus and DMA groups, the mRNA expression of TNF-α , TGF-β1 , and CTGF in DMA rats treated with human amniotic fluid stem cell-derived extracellular vesicles were significantly decreased at 4 weeks after induction, while the mRNA expression of IL-6 and Smad3 were significantly decreased 12 weeks. a P < 0.05 vs normal control (4 weeks and 12 weeks), b P < 0.05 vs diabetes mellitus (4 weeks and 12 weeks), c P < 0.05 vs diabetic atherosclerosis (4 weeks and 12 weeks). TNF: Tumor necrosis factor; IL: Interleukin; TGF: Transforming growth factor; CTGF: Connective tissue growth factor; DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles.

Article Snippet: The assay codes of TNF-α, IL-6, TGF-β1, Smad3, CTGF, and fibronectin were Rn01525859_g1, Rn01410330_m1, Rn00572010_m1, Rn00565331_m1, Rn01537279_g1 and Rn00569575_m1, respectively (Applied Biosystems, Oster City, CA, United States).

Techniques: Control, Expressing, Derivative Assay