smad3 signaling  (MedChemExpress)

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: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Iron inhibits the activation of SMAD3 in beige adipocytes. A Schematic diagram illustrating the protocol for ATAC-sequencing in in vitro differentiated beige adipocytes treated with FAC. B ATAC-seq signal distribution near the transcription start sites (TSS). C Pie chart showing the genomic distribution of all identified ATAC-seq peaks. D Motif enrichment analysis of differential ATAC-seq peaks (comparing FAC vs. CON). E Transcription factor footprint (TF) analysis for SMAD2::SMAD3 within differential ATAC-seq peaks. F Relative mRNA levels of Smad2 and Smad3 in CON and FAC-treated beige adipocytes. G , H Protein levels of phosphorylated and total SMAD2 and SMAD3 levels in CON and FAC-treated beige adipocytes. I , J Immunoblots phosphorylated SMAD3 (p-SMAD3), total SMAD3, UCP1, FABP4, and FTH1 in beige adipocytes treated with vehicle (CON), iron chelator deferoxamine (DFO), or DFO + FAC. Data in are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Activation Assay, Sequencing, In Vitro, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Inhibition of SMAD3 signaling promotes thermogenesis in beige adipocytes. A Representative Oil Red O staining of beige adipocytes treated with CON, FAC, SMAD3 inhibitor SB-431,542 (2µM) or SB-431,542 plus FAC. B Relative mRNA levels of thermogenic genes in cells treated as in ( A ). C - D Immunoblots of p-SMAD3, SMAD3, UCP1, FABP4, and FTH1 in cells treated as in ( A ). E Representative Oil Red O staining of beige adipocytes treated with CON or SMAD3 inhibitor SIS3. F Relative mRNA levels of thermogenic genes in cells treated as in ( E ). G , H Immunoblots of p-SMAD3, SMAD3, and UCP1 in cells treated as in ( E ). I , J Immunoblots of p-SMAD3/SMAD3 in iWAT from mice in CTRL and IED group (from Fig. ). K , L Immunoblots of p-SMAD3/SMAD3 in iWAT from vehicle and iron-injected mice (from Fig. ). Data in are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Inhibition, Staining, Western Blot, Injection

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: PSPC1 interacts with SMAD3 to regulate its activation and is involved in the regulation of iron on beiging of adipocytes. A Venn diagram illustrating the strategy to identify potential upstream regulators linking iron and SMAD3, integrating RNA-seq, ATAC-seq, published datasets ( GSE35011 , GSE22424 , GSE129573 ), and literature mining, leading to PSPC1. B ATAC-seq bedgraph panels of the Pspc1 locus showing the peak locations relative to the TSS. C Relative mRNA levels of Pspc1 in CON and FAC-treated beige adipocytes, measured by qPCR. D , E Immunoblots of PSPC1 and UCP1 protein levels in CON and FAC-treated cells. F Representative immunofluorescence images showing co-staining for DAPI (blue), SMAD3 (green), and PSPC1 (red) in beige adipocytes. Scale bar, 10 μm. G , H Co-immunoprecipitation (Co-IP) assays showing interaction between endogenous PSPC1 and SMAD3 in beige adipocyte. I , J Representative Oil Red O staining I and mRNA levels of adipogenic/thermogenic genes ( J ) in beige adipocytes transfected with control siRNA (siRNA-ctrl) or Pspc1 siRNA (siRNA-Pspc1). K Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FABP4 protein levels in cells treated as in ( I ) . L , M Representative Oil Red O staining and relative mRNA levels of adipogenic/thermogenic genes in beige adipocytes transduced with control (CON) or Pspc1 overexpression (PSPC1-OE) lentivirus, with or without subsequent FAC treatment. N Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FTH1 protein levels in cells treated as in ( L ). Data are presented as mean ± SEM. Scale bar, 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Transduction, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Iron-induced beiging of adipocytes and metabolic benefits is impaired by PSPC1 overexpression in mice. A Schematic diagram of the in vivo experiment. Mice fed with HFD in control (CTRL) or iron-enriched diet (IED) received iWAT injections of AAV expressing control vector (AAV-Vehicle) or Pspc1 (AAV- Pspc1 ) for a total of 10 weeks HFD feeding. B Body weight in CON and IED mice with AAV-Vehicle/Pspc1 at the end of the study. C Tissue weights of iWAT, eWAT, and BAT expressed as a percentage of total body weight. D Representative H&E staining of BAT, iWAT, and eWAT sections from the four groups. Scale bar, 50 μm. E , F GTT of mice in the four groups. G , H ITT of mice in the four groups. I - J VO2 during light and dark cycles with average values quantified. K Relative mRNA levels of Pspc1 and thermogenic/iron-related genes ( Ucp1 , Cidea , Pgc1a , Fth1 , Ftl1 ) in iWAT from CON and IED group with AAV-Vehicle/Pspc1. L Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FTH1 protein levels in iWAT. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Over Expression, In Vivo, Control, Expressing, Plasmid Preparation, Staining, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Iron inhibits the activation of SMAD3 in beige adipocytes. A Schematic diagram illustrating the protocol for ATAC-sequencing in in vitro differentiated beige adipocytes treated with FAC. B ATAC-seq signal distribution near the transcription start sites (TSS). C Pie chart showing the genomic distribution of all identified ATAC-seq peaks. D Motif enrichment analysis of differential ATAC-seq peaks (comparing FAC vs. CON). E Transcription factor footprint (TF) analysis for SMAD2::SMAD3 within differential ATAC-seq peaks. F Relative mRNA levels of Smad2 and Smad3 in CON and FAC-treated beige adipocytes. G , H Protein levels of phosphorylated and total SMAD2 and SMAD3 levels in CON and FAC-treated beige adipocytes. I , J Immunoblots phosphorylated SMAD3 (p-SMAD3), total SMAD3, UCP1, FABP4, and FTH1 in beige adipocytes treated with vehicle (CON), iron chelator deferoxamine (DFO), or DFO + FAC. Data in are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Activation Assay, Sequencing, In Vitro, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Inhibition of SMAD3 signaling promotes thermogenesis in beige adipocytes. A Representative Oil Red O staining of beige adipocytes treated with CON, FAC, SMAD3 inhibitor SB-431,542 (2µM) or SB-431,542 plus FAC. B Relative mRNA levels of thermogenic genes in cells treated as in ( A ). C - D Immunoblots of p-SMAD3, SMAD3, UCP1, FABP4, and FTH1 in cells treated as in ( A ). E Representative Oil Red O staining of beige adipocytes treated with CON or SMAD3 inhibitor SIS3. F Relative mRNA levels of thermogenic genes in cells treated as in ( E ). G , H Immunoblots of p-SMAD3, SMAD3, and UCP1 in cells treated as in ( E ). I , J Immunoblots of p-SMAD3/SMAD3 in iWAT from mice in CTRL and IED group (from Fig. ). K , L Immunoblots of p-SMAD3/SMAD3 in iWAT from vehicle and iron-injected mice (from Fig. ). Data in are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Inhibition, Staining, Western Blot, Injection

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: PSPC1 interacts with SMAD3 to regulate its activation and is involved in the regulation of iron on beiging of adipocytes. A Venn diagram illustrating the strategy to identify potential upstream regulators linking iron and SMAD3, integrating RNA-seq, ATAC-seq, published datasets ( GSE35011 , GSE22424 , GSE129573 ), and literature mining, leading to PSPC1. B ATAC-seq bedgraph panels of the Pspc1 locus showing the peak locations relative to the TSS. C Relative mRNA levels of Pspc1 in CON and FAC-treated beige adipocytes, measured by qPCR. D , E Immunoblots of PSPC1 and UCP1 protein levels in CON and FAC-treated cells. F Representative immunofluorescence images showing co-staining for DAPI (blue), SMAD3 (green), and PSPC1 (red) in beige adipocytes. Scale bar, 10 μm. G , H Co-immunoprecipitation (Co-IP) assays showing interaction between endogenous PSPC1 and SMAD3 in beige adipocyte. I , J Representative Oil Red O staining I and mRNA levels of adipogenic/thermogenic genes ( J ) in beige adipocytes transfected with control siRNA (siRNA-ctrl) or Pspc1 siRNA (siRNA-Pspc1). K Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FABP4 protein levels in cells treated as in ( I ) . L , M Representative Oil Red O staining and relative mRNA levels of adipogenic/thermogenic genes in beige adipocytes transduced with control (CON) or Pspc1 overexpression (PSPC1-OE) lentivirus, with or without subsequent FAC treatment. N Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FTH1 protein levels in cells treated as in ( L ). Data are presented as mean ± SEM. Scale bar, 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Transduction, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Iron-induced beiging of adipocytes and metabolic benefits is impaired by PSPC1 overexpression in mice. A Schematic diagram of the in vivo experiment. Mice fed with HFD in control (CTRL) or iron-enriched diet (IED) received iWAT injections of AAV expressing control vector (AAV-Vehicle) or Pspc1 (AAV- Pspc1 ) for a total of 10 weeks HFD feeding. B Body weight in CON and IED mice with AAV-Vehicle/Pspc1 at the end of the study. C Tissue weights of iWAT, eWAT, and BAT expressed as a percentage of total body weight. D Representative H&E staining of BAT, iWAT, and eWAT sections from the four groups. Scale bar, 50 μm. E , F GTT of mice in the four groups. G , H ITT of mice in the four groups. I - J VO2 during light and dark cycles with average values quantified. K Relative mRNA levels of Pspc1 and thermogenic/iron-related genes ( Ucp1 , Cidea , Pgc1a , Fth1 , Ftl1 ) in iWAT from CON and IED group with AAV-Vehicle/Pspc1. L Immunoblots of PSPC1, p-SMAD3, SMAD3, UCP1, and FTH1 protein levels in iWAT. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: For specific inhibition of SMAD3, 0.5μM specific inhibitor of Smad3 (SIS3) (MCE, HY-13013) or the inhibitor SB-431,542 (2μM) was added in the medium during the differentiation.

Techniques: Over Expression, In Vivo, Control, Expressing, Plasmid Preparation, Staining, Western Blot