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stat3 inhibitor s3i 201 (Santa Cruz Biotechnology)

Name: stat3 inhibitor s3i 201
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Santa Cruz Biotechnology stat3 inhibitor s3i 201

a Immunoblot analysis was performed to assess Pellino1, <t>STAT3,</t> and p-STAT3 (Y705) protein levels in lysates of intestinal macrophages isolated from WT and Pellino1-mKO male mice in normal, acute 1.5% DSS and AOM/DSS groups. Actin was used as a loading control. b Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of BMDMs from WT and Pellino1-mKO mice. BMDMs were stimulated with 100 ng/mL LPS for the indicated time. Actin was used as a loading control. c GST pulldown assay was performed by incubating RAW cell lysates treated with or without 100 ng/mL LPS for 6 h, along with GST or GST-STAT3. d Co-immunization (Co-IP) assays were performed using HEK293T cells transfected with expression vectors for Myc, Myc-tagged Pellino1 (Myc-Pellino1), and Flag-tagged STAT3 (Flag-STAT3). Cellular extracts were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody. e GST (black arrow) or GST-STAT3 (blue arrow) protein was incubated with purified His-Pellino1 and subjected to immunoblotting using anti-Pellino1 and anti-GST antibodies. CS: Coomassie brilliant blue staining. f WT and Pellino1-mKO BMDMs were stimulated with 100 ng/mL LPS for 6 h. Immunofluorescence staining of Pellino1 (red), p-STAT3 (green), and DAPI (blue). DAPI was used to stain the nuclei. Scale bar = 10 μm. g GST pulldown assay was performed using purified GST or GST-Pellino1 protein with RAW cell lysates. RAW cells were stimulated with 100 ng/mL LPS for 6 h. h For IP assays, LPS-untreated RAW cells and RAW cells treated with 100 ng/mL LPS for 6 h were lysed. Subsequently, immunoprecipitation was performed using an anti-Pellino1 antibody, followed by immunoblotting with an anti-STAT3 antibody. i Co-IP assays were performed using HEK293T cells transfected with expression vectors for Myc-Pellino1, Flag-STAT3 WT, and Flag-STAT3 dominant negative (Flag-STAT3 DN; Flag-STAT3 Y705F). Cellular extracts were subjected to immunoprecipitation using an anti-Myc antibody and subsequently immunoblotted with anti-Flag and anti-Myc antibodies. Source data are provided as a Source Data file.

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1) Product Images from "The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development"

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

Journal: Nature Communications

doi: 10.1038/s41467-025-56440-6

a Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of intestinal macrophages isolated from WT and Pellino1-mKO male mice in normal, acute 1.5% DSS and AOM/DSS groups. Actin was used as a loading control. b Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of BMDMs from WT and Pellino1-mKO mice. BMDMs were stimulated with 100 ng/mL LPS for the indicated time. Actin was used as a loading control. c GST pulldown assay was performed by incubating RAW cell lysates treated with or without 100 ng/mL LPS for 6 h, along with GST or GST-STAT3. d Co-immunization (Co-IP) assays were performed using HEK293T cells transfected with expression vectors for Myc, Myc-tagged Pellino1 (Myc-Pellino1), and Flag-tagged STAT3 (Flag-STAT3). Cellular extracts were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody. e GST (black arrow) or GST-STAT3 (blue arrow) protein was incubated with purified His-Pellino1 and subjected to immunoblotting using anti-Pellino1 and anti-GST antibodies. CS: Coomassie brilliant blue staining. f WT and Pellino1-mKO BMDMs were stimulated with 100 ng/mL LPS for 6 h. Immunofluorescence staining of Pellino1 (red), p-STAT3 (green), and DAPI (blue). DAPI was used to stain the nuclei. Scale bar = 10 μm. g GST pulldown assay was performed using purified GST or GST-Pellino1 protein with RAW cell lysates. RAW cells were stimulated with 100 ng/mL LPS for 6 h. h For IP assays, LPS-untreated RAW cells and RAW cells treated with 100 ng/mL LPS for 6 h were lysed. Subsequently, immunoprecipitation was performed using an anti-Pellino1 antibody, followed by immunoblotting with an anti-STAT3 antibody. i Co-IP assays were performed using HEK293T cells transfected with expression vectors for Myc-Pellino1, Flag-STAT3 WT, and Flag-STAT3 dominant negative (Flag-STAT3 DN; Flag-STAT3 Y705F). Cellular extracts were subjected to immunoprecipitation using an anti-Myc antibody and subsequently immunoblotted with anti-Flag and anti-Myc antibodies. Source data are provided as a Source Data file.

Figure Legend Snippet: a Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of intestinal macrophages isolated from WT and Pellino1-mKO male mice in normal, acute 1.5% DSS and AOM/DSS groups. Actin was used as a loading control. b Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of BMDMs from WT and Pellino1-mKO mice. BMDMs were stimulated with 100 ng/mL LPS for the indicated time. Actin was used as a loading control. c GST pulldown assay was performed by incubating RAW cell lysates treated with or without 100 ng/mL LPS for 6 h, along with GST or GST-STAT3. d Co-immunization (Co-IP) assays were performed using HEK293T cells transfected with expression vectors for Myc, Myc-tagged Pellino1 (Myc-Pellino1), and Flag-tagged STAT3 (Flag-STAT3). Cellular extracts were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody. e GST (black arrow) or GST-STAT3 (blue arrow) protein was incubated with purified His-Pellino1 and subjected to immunoblotting using anti-Pellino1 and anti-GST antibodies. CS: Coomassie brilliant blue staining. f WT and Pellino1-mKO BMDMs were stimulated with 100 ng/mL LPS for 6 h. Immunofluorescence staining of Pellino1 (red), p-STAT3 (green), and DAPI (blue). DAPI was used to stain the nuclei. Scale bar = 10 μm. g GST pulldown assay was performed using purified GST or GST-Pellino1 protein with RAW cell lysates. RAW cells were stimulated with 100 ng/mL LPS for 6 h. h For IP assays, LPS-untreated RAW cells and RAW cells treated with 100 ng/mL LPS for 6 h were lysed. Subsequently, immunoprecipitation was performed using an anti-Pellino1 antibody, followed by immunoblotting with an anti-STAT3 antibody. i Co-IP assays were performed using HEK293T cells transfected with expression vectors for Myc-Pellino1, Flag-STAT3 WT, and Flag-STAT3 dominant negative (Flag-STAT3 DN; Flag-STAT3 Y705F). Cellular extracts were subjected to immunoprecipitation using an anti-Myc antibody and subsequently immunoblotted with anti-Flag and anti-Myc antibodies. Source data are provided as a Source Data file.

Techniques Used: Western Blot, Isolation, Control, GST Pulldown Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Incubation, Purification, Staining, Immunofluorescence, Dominant Negative Mutation

a HEK293T cells were co-transfected with Myc-tagged Pellino1 WT (full-length; FL) or Myc-tagged Pellino1 with a C-terminal deletion (ΔC), Flag-STAT3, and HA-Ub expression plasmids. At 48 h after transfection, cells were treated with MG132 for 5 h, followed by an additional treatment with 1 µg/mL LPS for 30 min. Immunoprecipitation was performed using an anti-Ub antibody. Samples were analyzed by immunoblotting with an anti-STAT3 antibody. The graphic representation depicts Myc-Pellino1 FL and Pellino1 truncation mutants (ΔC), where FHA represents the FHA domain and RING signifies the RING-like domain. b In vitro ubiquitination assay of GST-STAT3 was performed using recombinant E1 (UBE1), recombinant E2 (Ubc13), HA-Ub, and His-Pellino1. Mixtures were incubated at 37 °C in an assay buffer containing ATP for 2 h. Immunoblot analysis was performed using anti-STAT3 and anti-Ub antibodies. c HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48 Ub, and HA-K63 Ub. At 48 h post-transfection, cells were treated with MG132 for 5 h and subsequently stimulated with 1 μg/mL LPS for 30 min. Cell lysates were immunoprecipitated with an anti-Ub antibody and immunoblotted with an anti-STAT3 antibody. d HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48R Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( c ). e Intestinal macrophages were isolated from WT and Pellino1-mKO male mice of normal and acute 1.5% DSS groups. Cells were lysed and immunoprecipitated using an anti-Ub antibody. Immunoprecipitated samples were then analyzed by immunoblotting with an anti-STAT3. f WT and Pellino1-mKO BMDMs were treated with 100 ng/mL LPS for 3 h and then lysed. Cell lysates were immunoprecipitated with an anti-p-STAT3 antibody and immunoblotted with an anti-Ub antibody. Source data are provided as a Source Data file.

Figure Legend Snippet: a HEK293T cells were co-transfected with Myc-tagged Pellino1 WT (full-length; FL) or Myc-tagged Pellino1 with a C-terminal deletion (ΔC), Flag-STAT3, and HA-Ub expression plasmids. At 48 h after transfection, cells were treated with MG132 for 5 h, followed by an additional treatment with 1 µg/mL LPS for 30 min. Immunoprecipitation was performed using an anti-Ub antibody. Samples were analyzed by immunoblotting with an anti-STAT3 antibody. The graphic representation depicts Myc-Pellino1 FL and Pellino1 truncation mutants (ΔC), where FHA represents the FHA domain and RING signifies the RING-like domain. b In vitro ubiquitination assay of GST-STAT3 was performed using recombinant E1 (UBE1), recombinant E2 (Ubc13), HA-Ub, and His-Pellino1. Mixtures were incubated at 37 °C in an assay buffer containing ATP for 2 h. Immunoblot analysis was performed using anti-STAT3 and anti-Ub antibodies. c HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48 Ub, and HA-K63 Ub. At 48 h post-transfection, cells were treated with MG132 for 5 h and subsequently stimulated with 1 μg/mL LPS for 30 min. Cell lysates were immunoprecipitated with an anti-Ub antibody and immunoblotted with an anti-STAT3 antibody. d HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48R Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( c ). e Intestinal macrophages were isolated from WT and Pellino1-mKO male mice of normal and acute 1.5% DSS groups. Cells were lysed and immunoprecipitated using an anti-Ub antibody. Immunoprecipitated samples were then analyzed by immunoblotting with an anti-STAT3. f WT and Pellino1-mKO BMDMs were treated with 100 ng/mL LPS for 3 h and then lysed. Cell lysates were immunoprecipitated with an anti-p-STAT3 antibody and immunoblotted with an anti-Ub antibody. Source data are provided as a Source Data file.

Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Proteomics, Recombinant, Incubation, Isolation

$a (Left) Immunoblotting of STAT3 and p-STAT3 (Y705) in WT and Pellino1-mKO BMDMs treated with 12.5 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 100 ng/mL LPS for 3 h before CHX treatment. (Right) Levels of phosphorylated STAT3 were quantified by densitometric analysis of immunoblots using ImageJ ( n = 4). b HEK293T cells were transfected with Myc or Myc-Pellino1. After 48 h of co-transfection, cells were treated with 100 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 1 μg/mL LPS for 30 min before CHX treatment. c HEK293T cells were co-transfected with Myc-Pellino1, HA-Ub WT, HA-K63 Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( b ). d WT and Pellino1-mKO BMDMs were fractionated after 3 h of treatment with 100 ng/mL LPS. Immunoblot analysis was performed to assess p-STAT3 (Y705) protein levels in the cytoplasm and nucleus. CF cytosolic fraction, NF nuclear fraction. e ChIP-qRT-PCR analysis was performed using anti-STAT3 or control IgG with WT and Pellino1-mKO BMDMs as shown in the figure. BMDMs were stimulated with 100 ng/mL LPS for 6 h. ChIP data are presented as relative enrichment normalized to input in WT or Pellino1-mKO BMDMs ( n = 3). f MMP9 , BCL2 , and IL6 mRNA levels in WT and Pellino1 deficient BMDMs ( n = 3) were quantified with qRT-PCR and normalized against GAPDH expression. g MMP9 , BCL2 , IL6 , and VEGF mRNA levels in intestinal macrophages from WT and Pellino1-mKO male mice of normal, acute 1.5% DSS, and AOM/DSS groups ( n = 4) were quantified with qRT-PCR and normalized against GAPDH expression. Data were represented as mean ± SD in ( a , e – g ). All statistical comparisons were made using a two-tailed Student’s t -test. Source data are provided as a Source Data file.$
Figure Legend Snippet: a (Left) Immunoblotting of STAT3 and p-STAT3 (Y705) in WT and Pellino1-mKO BMDMs treated with 12.5 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 100 ng/mL LPS for 3 h before CHX treatment. (Right) Levels of phosphorylated STAT3 were quantified by densitometric analysis of immunoblots using ImageJ ( n = 4). b HEK293T cells were transfected with Myc or Myc-Pellino1. After 48 h of co-transfection, cells were treated with 100 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 1 μg/mL LPS for 30 min before CHX treatment. c HEK293T cells were co-transfected with Myc-Pellino1, HA-Ub WT, HA-K63 Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( b ). d WT and Pellino1-mKO BMDMs were fractionated after 3 h of treatment with 100 ng/mL LPS. Immunoblot analysis was performed to assess p-STAT3 (Y705) protein levels in the cytoplasm and nucleus. CF cytosolic fraction, NF nuclear fraction. e ChIP-qRT-PCR analysis was performed using anti-STAT3 or control IgG with WT and Pellino1-mKO BMDMs as shown in the figure. BMDMs were stimulated with 100 ng/mL LPS for 6 h. ChIP data are presented as relative enrichment normalized to input in WT or Pellino1-mKO BMDMs ( n = 3). f MMP9 , BCL2 , and IL6 mRNA levels in WT and Pellino1 deficient BMDMs ( n = 3) were quantified with qRT-PCR and normalized against GAPDH expression. g MMP9 , BCL2 , IL6 , and VEGF mRNA levels in intestinal macrophages from WT and Pellino1-mKO male mice of normal, acute 1.5% DSS, and AOM/DSS groups ( n = 4) were quantified with qRT-PCR and normalized against GAPDH expression. Data were represented as mean ± SD in ( a , e – g ). All statistical comparisons were made using a two-tailed Student’s t -test. Source data are provided as a Source Data file.

Techniques Used: Western Blot, Transfection, Cotransfection, Quantitative RT-PCR, Control, Expressing, Two Tailed Test

The Pellino1-STAT3 pathway is crucial in the pathogenesis of colitis and CAC through the K63-linked ubiquitination of STAT3.

Figure Legend Snippet: The Pellino1-STAT3 pathway is crucial in the pathogenesis of colitis and CAC through the K63-linked ubiquitination of STAT3.

Techniques Used: Ubiquitin Proteomics

Image Search Results

A The standard chemical structure of L-cystine. B Cell viability measured by CCK-8 assay. C IL-6 mRNA and protein levels analyzed by qRT-PCR and Western blot. D IL-6 concentration in cell supernatant quantified by ELISA. E ROS generation assessed via flow cytometry. F Intracellular Fe²⁺ levels. G 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. H , I MDA, GSH, and SOD levels. J Western blot analysis of STAT3 phosphorylation and NCOA4/FTH1 protein levels. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: L-cystine alleviates necrotizing enterocolitis by regulating ferroptosis and Th17 cell differentiation via the IL-6/STAT3 pathway

doi: 10.1038/s42003-025-09438-1

Figure Lengend Snippet: A The standard chemical structure of L-cystine. B Cell viability measured by CCK-8 assay. C IL-6 mRNA and protein levels analyzed by qRT-PCR and Western blot. D IL-6 concentration in cell supernatant quantified by ELISA. E ROS generation assessed via flow cytometry. F Intracellular Fe²⁺ levels. G 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. H , I MDA, GSH, and SOD levels. J Western blot analysis of STAT3 phosphorylation and NCOA4/FTH1 protein levels. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For therapeutic intervention, pups received daily oral L-cystine (200 mg/mL in saline solution; C6H12N2O4S2, 56-89-3, MCE, Monmouth Junction, NJ, USA) and intraperitoneal injections of the STAT3 inhibitor S3I-201 (0.5 mg/mL in DMSO and 20% cyclodextrin; 501919-59-1, MCE) twice weekly .

Techniques: CCK-8 Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Immunofluorescence, Phospho-proteomics

A Cell viability assessed by CCK-8. B Fe²⁺ levels. C 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. D ROS generation via flow cytometry. E MDA, GSH, and SOD levels. F STAT3 phosphorylation and NCOA4/FTH1 levels using Western blot. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: L-cystine alleviates necrotizing enterocolitis by regulating ferroptosis and Th17 cell differentiation via the IL-6/STAT3 pathway

doi: 10.1038/s42003-025-09438-1

Figure Lengend Snippet: A Cell viability assessed by CCK-8. B Fe²⁺ levels. C 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. D ROS generation via flow cytometry. E MDA, GSH, and SOD levels. F STAT3 phosphorylation and NCOA4/FTH1 levels using Western blot. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Phospho-proteomics, Western Blot

A Cell viability measured by CCK-8. B ROS generation via flow cytometry. C Fe²⁺ levels. D 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. E MDA, GSH, and SOD levels. F IL-6 concentration in supernatant by ELISA. G STAT3 phosphorylation and NCOA4/FTH1 levels using Western blot. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: L-cystine alleviates necrotizing enterocolitis by regulating ferroptosis and Th17 cell differentiation via the IL-6/STAT3 pathway

doi: 10.1038/s42003-025-09438-1

Figure Lengend Snippet: A Cell viability measured by CCK-8. B ROS generation via flow cytometry. C Fe²⁺ levels. D 4-HNE expression detected by immunofluorescence. Scale bar = 25 μm. E MDA, GSH, and SOD levels. F IL-6 concentration in supernatant by ELISA. G STAT3 phosphorylation and NCOA4/FTH1 levels using Western blot. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot

A Flow cytometry sorting of native CD4 + T cells from patient peripheral blood. B , C Native CD4 + T cells treated with NCM-460-conditioned medium (CM) and anti-IL-6 antibody (900 nM) for 48 h. Flow cytometry analysis of Treg/Th17 populations and ratios. D , E Native CD4 + T cells treated with gradient concentrations of recombinant IL-6 (rIL-6). Flow cytometry analysis of Treg/Th17 populations and ratios. F , G Native CD4 + T cells treated with NCM-460-CM, rIL-6 (20 ng/mL), and STAT3 inhibitor S3I-201 (50 µM) for 48 h. Flow cytometry analysis of Treg/Th17 populations and ratios. H Western blot analysis of STAT3 phosphorylation. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: L-cystine alleviates necrotizing enterocolitis by regulating ferroptosis and Th17 cell differentiation via the IL-6/STAT3 pathway

doi: 10.1038/s42003-025-09438-1

Figure Lengend Snippet: A Flow cytometry sorting of native CD4 + T cells from patient peripheral blood. B , C Native CD4 + T cells treated with NCM-460-conditioned medium (CM) and anti-IL-6 antibody (900 nM) for 48 h. Flow cytometry analysis of Treg/Th17 populations and ratios. D , E Native CD4 + T cells treated with gradient concentrations of recombinant IL-6 (rIL-6). Flow cytometry analysis of Treg/Th17 populations and ratios. F , G Native CD4 + T cells treated with NCM-460-CM, rIL-6 (20 ng/mL), and STAT3 inhibitor S3I-201 (50 µM) for 48 h. Flow cytometry analysis of Treg/Th17 populations and ratios. H Western blot analysis of STAT3 phosphorylation. n = 3 biologically independent samples. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Flow Cytometry, Recombinant, Western Blot, Phospho-proteomics

A MDA, GSH, and SOD levels. B Flow cytometry analysis of ROS generation. C Fe²⁺ levels. D Mitochondrial morphology in colon tissues using TEM. Scale bar = 500 nm. E , F Immunofluorescence showing 4-HNE expression in colon tissues. Scale bar = 25 μm. G Western blot analysis of STAT3 phosphorylation and NCOA4/FTH1 levels. n = 3 animals (both sexes). Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: L-cystine alleviates necrotizing enterocolitis by regulating ferroptosis and Th17 cell differentiation via the IL-6/STAT3 pathway

doi: 10.1038/s42003-025-09438-1

Figure Lengend Snippet: A MDA, GSH, and SOD levels. B Flow cytometry analysis of ROS generation. C Fe²⁺ levels. D Mitochondrial morphology in colon tissues using TEM. Scale bar = 500 nm. E , F Immunofluorescence showing 4-HNE expression in colon tissues. Scale bar = 25 μm. G Western blot analysis of STAT3 phosphorylation and NCOA4/FTH1 levels. n = 3 animals (both sexes). Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Flow Cytometry, Immunofluorescence, Expressing, Western Blot, Phospho-proteomics

IL-21 induces STAT3—Blimp-1—GATA3 expression, and JAK/STAT3 inhibitors reduce this effect. (A) Flow cytometry analysis of the expression proportion of phosphorylated STAT3 (pSTAT3) in γδ T cells and Vδ2 T cells, induced by IL-21. (B) Flow cytometry analysis of the expression proportion of Blimp-1 in γδ T cells and Vδ2 T cells, induced by IL-21. (C) Comparison of the expression levels of pSTAT3, Blimp-1, and GATA3 proteins between the control group and the IL-21-treated experimental group. Paired t-tests were used to compare the same samples between the control and experimental conditions. (D-F) Flow cytometry analysis of the effect of JAK/STAT3 inhibitors on the expression of pSTAT3 (D) , Blimp-1 (E) , GATA3 (F) , induced by IL-21 in γδ T cells and Vδ2 T cells. Tofa 1 , Tofacitinib (10 nM); Tofa 2 , Tofacitinib (100 nM); Bari, Baricitinib (300 nM); S3I, STAT3 inhibitor S3I-201 (10 μM). IPP, isopentenyl pyrophosphate; +IL-21, experimental group with IL-21 treatment; Control, control group without IL-21. *P < 0.05, **P < 0.01.

Journal: Frontiers in Immunology

Article Title: Polarization of Vδ2 T cells to a Th2-like phenotype promotes plasmablast differentiation and possesses pro-fibrotic properties in IgG4-related disease

doi: 10.3389/fimmu.2025.1550405

Figure Lengend Snippet: IL-21 induces STAT3—Blimp-1—GATA3 expression, and JAK/STAT3 inhibitors reduce this effect. (A) Flow cytometry analysis of the expression proportion of phosphorylated STAT3 (pSTAT3) in γδ T cells and Vδ2 T cells, induced by IL-21. (B) Flow cytometry analysis of the expression proportion of Blimp-1 in γδ T cells and Vδ2 T cells, induced by IL-21. (C) Comparison of the expression levels of pSTAT3, Blimp-1, and GATA3 proteins between the control group and the IL-21-treated experimental group. Paired t-tests were used to compare the same samples between the control and experimental conditions. (D-F) Flow cytometry analysis of the effect of JAK/STAT3 inhibitors on the expression of pSTAT3 (D) , Blimp-1 (E) , GATA3 (F) , induced by IL-21 in γδ T cells and Vδ2 T cells. Tofa 1 , Tofacitinib (10 nM); Tofa 2 , Tofacitinib (100 nM); Bari, Baricitinib (300 nM); S3I, STAT3 inhibitor S3I-201 (10 μM). IPP, isopentenyl pyrophosphate; +IL-21, experimental group with IL-21 treatment; Control, control group without IL-21. *P < 0.05, **P < 0.01.

Article Snippet: To investigate the role of IL-21 signaling in Vδ2 T cell differentiation, γδ T cells were stimulated with IL-21 (100 ng/ml, PeproTech) or treated with JAK/STAT3 pathway inhibitors, including Baricitinib (300 nM), Tofacitinib (10 nM and 100 nM), and STAT3 inhibitor S3I-201 (NSC 74859) (Selleck).

Techniques: Expressing, Flow Cytometry, Comparison, Control

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of intestinal macrophages isolated from WT and Pellino1-mKO male mice in normal, acute 1.5% DSS and AOM/DSS groups. Actin was used as a loading control. b Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of BMDMs from WT and Pellino1-mKO mice. BMDMs were stimulated with 100 ng/mL LPS for the indicated time. Actin was used as a loading control. c GST pulldown assay was performed by incubating RAW cell lysates treated with or without 100 ng/mL LPS for 6 h, along with GST or GST-STAT3. d Co-immunization (Co-IP) assays were performed using HEK293T cells transfected with expression vectors for Myc, Myc-tagged Pellino1 (Myc-Pellino1), and Flag-tagged STAT3 (Flag-STAT3). Cellular extracts were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody. e GST (black arrow) or GST-STAT3 (blue arrow) protein was incubated with purified His-Pellino1 and subjected to immunoblotting using anti-Pellino1 and anti-GST antibodies. CS: Coomassie brilliant blue staining. f WT and Pellino1-mKO BMDMs were stimulated with 100 ng/mL LPS for 6 h. Immunofluorescence staining of Pellino1 (red), p-STAT3 (green), and DAPI (blue). DAPI was used to stain the nuclei. Scale bar = 10 μm. g GST pulldown assay was performed using purified GST or GST-Pellino1 protein with RAW cell lysates. RAW cells were stimulated with 100 ng/mL LPS for 6 h. h For IP assays, LPS-untreated RAW cells and RAW cells treated with 100 ng/mL LPS for 6 h were lysed. Subsequently, immunoprecipitation was performed using an anti-Pellino1 antibody, followed by immunoblotting with an anti-STAT3 antibody. i Co-IP assays were performed using HEK293T cells transfected with expression vectors for Myc-Pellino1, Flag-STAT3 WT, and Flag-STAT3 dominant negative (Flag-STAT3 DN; Flag-STAT3 Y705F). Cellular extracts were subjected to immunoprecipitation using an anti-Myc antibody and subsequently immunoblotted with anti-Flag and anti-Myc antibodies. Source data are provided as a Source Data file.

Article Snippet: STAT3 inhibitor S3I-201 (CAS 501919-59-1) was purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Isolation, Control, GST Pulldown Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Incubation, Purification, Staining, Immunofluorescence, Dominant Negative Mutation

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a HEK293T cells were co-transfected with Myc-tagged Pellino1 WT (full-length; FL) or Myc-tagged Pellino1 with a C-terminal deletion (ΔC), Flag-STAT3, and HA-Ub expression plasmids. At 48 h after transfection, cells were treated with MG132 for 5 h, followed by an additional treatment with 1 µg/mL LPS for 30 min. Immunoprecipitation was performed using an anti-Ub antibody. Samples were analyzed by immunoblotting with an anti-STAT3 antibody. The graphic representation depicts Myc-Pellino1 FL and Pellino1 truncation mutants (ΔC), where FHA represents the FHA domain and RING signifies the RING-like domain. b In vitro ubiquitination assay of GST-STAT3 was performed using recombinant E1 (UBE1), recombinant E2 (Ubc13), HA-Ub, and His-Pellino1. Mixtures were incubated at 37 °C in an assay buffer containing ATP for 2 h. Immunoblot analysis was performed using anti-STAT3 and anti-Ub antibodies. c HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48 Ub, and HA-K63 Ub. At 48 h post-transfection, cells were treated with MG132 for 5 h and subsequently stimulated with 1 μg/mL LPS for 30 min. Cell lysates were immunoprecipitated with an anti-Ub antibody and immunoblotted with an anti-STAT3 antibody. d HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48R Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( c ). e Intestinal macrophages were isolated from WT and Pellino1-mKO male mice of normal and acute 1.5% DSS groups. Cells were lysed and immunoprecipitated using an anti-Ub antibody. Immunoprecipitated samples were then analyzed by immunoblotting with an anti-STAT3. f WT and Pellino1-mKO BMDMs were treated with 100 ng/mL LPS for 3 h and then lysed. Cell lysates were immunoprecipitated with an anti-p-STAT3 antibody and immunoblotted with an anti-Ub antibody. Source data are provided as a Source Data file.

Article Snippet: STAT3 inhibitor S3I-201 (CAS 501919-59-1) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Proteomics, Recombinant, Incubation, Isolation

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a (Left) Immunoblotting of STAT3 and p-STAT3 (Y705) in WT and Pellino1-mKO BMDMs treated with 12.5 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 100 ng/mL LPS for 3 h before CHX treatment. (Right) Levels of phosphorylated STAT3 were quantified by densitometric analysis of immunoblots using ImageJ ( n = 4). b HEK293T cells were transfected with Myc or Myc-Pellino1. After 48 h of co-transfection, cells were treated with 100 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 1 μg/mL LPS for 30 min before CHX treatment. c HEK293T cells were co-transfected with Myc-Pellino1, HA-Ub WT, HA-K63 Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( b ). d WT and Pellino1-mKO BMDMs were fractionated after 3 h of treatment with 100 ng/mL LPS. Immunoblot analysis was performed to assess p-STAT3 (Y705) protein levels in the cytoplasm and nucleus. CF cytosolic fraction, NF nuclear fraction. e ChIP-qRT-PCR analysis was performed using anti-STAT3 or control IgG with WT and Pellino1-mKO BMDMs as shown in the figure. BMDMs were stimulated with 100 ng/mL LPS for 6 h. ChIP data are presented as relative enrichment normalized to input in WT or Pellino1-mKO BMDMs ( n = 3). f MMP9 , BCL2 , and IL6 mRNA levels in WT and Pellino1 deficient BMDMs ( n = 3) were quantified with qRT-PCR and normalized against GAPDH expression. g MMP9 , BCL2 , IL6 , and VEGF mRNA levels in intestinal macrophages from WT and Pellino1-mKO male mice of normal, acute 1.5% DSS, and AOM/DSS groups ( n = 4) were quantified with qRT-PCR and normalized against GAPDH expression. Data were represented as mean ± SD in ( a , e – g ). All statistical comparisons were made using a two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: STAT3 inhibitor S3I-201 (CAS 501919-59-1) was purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Cotransfection, Quantitative RT-PCR, Control, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: The Pellino1-STAT3 pathway is crucial in the pathogenesis of colitis and CAC through the K63-linked ubiquitination of STAT3.

Article Snippet: STAT3 inhibitor S3I-201 (CAS 501919-59-1) was purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics

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