ac stat3 cell signaling  (Cell Signaling Technology Inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the HLA-E–NKG2A axis in combination with MS-275 enhances NK cell-based immunotherapy against DMG

doi: 10.1186/s13046-025-03390-y

Figure Lengend Snippet: MS-275 enhances HLA-E expression through acetylation of STAT3. A A Venn diagram showing the analysis of the KnockTF, ENCODE, and ChIP-Atlas databases revealed 56, 113, and 422 HLA-E transcription factors, respectively. Intersection analysis identified 11 most likely HLA-E transcription factors, including JUND, NFYA, NFYB, RELA, RFX5, RNF2, STAT1, STAT2, STAT3, STAT5A and TRIM28. B - C Lollipop chart showing correlation analysis between 11 potential transcription factors (JUND, NFYA, NFYB, RELA, RFX5, RNF2, STAT1, STAT2, STAT3, STAT5A, and TRIM28) and HLA-E expression in BSG ( B ) or DMG ( C ) patients. The point size represents the correlation coefficient. D Integrated Genomics Viewer (IGV) screenshot showing the results of STAT3 ChIP-seq in peaks at the genomic regions of HLA-E. E Analysis of the JASPAR database showing the potential binding region of STAT3 to the HLA-E promoter. F The protein expression of HLA-E and total STAT3 in TT150630 cells expressing sh-NC or sh-STAT3 was detected via Western blotting. β-Actin was used as the internal control (left). The quantified results are presented in the plot (right). Statistical significance was assessed via one-way ANOVA with ** p < 0.01 and *** p < 0.001. G HLA-E expression on the cell surface of TT150630 cells expressing sh-NC or sh-STAT3 was detected via flow cytometry. The quantification of the results is shown on the right. Statistical significance was assessed via one-way ANOVA with **** p < 0.0001. H TT150630 cells treated with MS-275 (1 μM) or DMSO for 2 days were analyzed for STAT3 recruitment to the HLA-E promoter via ChIP analysis. The ChIP results are shown as the fold change relative to the IgG control. Statistical significance was assessed via Student’s t test, with ** p < 0.01 and *** p < 0.001. I The protein expression of HLA-E, total STAT3, p-STAT3 (S727), p-STAT3 (Y705), and ac-STAT3 (K685) in TT150630 and TT190326 cells treated with 1 μM MS-275 for 2 days was detected via Western blotting. β-Actin was used as the internal control. J The protein expression of HLA-E, total STAT3, p-STAT3 (S727), and p-STAT3 (Y705) in TT150630 and TT190326 cells treated with 1 μM Stattic for 2 days was detected via Western blotting. β-Actin was used as the internal control. K The protein expression of HLA-E, total STAT3, and ac-STAT3 (K685) in TT150630 cells or TT190326 cells expressing sh-NC or sh-STAT3 treated with DMOS or 1 μM MS-275 for 2 days was detected via Western blotting. β-Actin was used as the internal control

Article Snippet: The membranes were probed for STAT3 (CST, Cat# 9139), ac-STAT3 K685 (CST, Cat# 2523), p-STAT3 S727 (CST, Cat# 94994), p-STAT3 Y705 (CST, Cat# 9145), HLA-E (proteintech, Cat# 66530-1-Ig), and β-actin (Abcam, Cat# ab20272).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Western Blot, Control, Flow Cytometry

Journal: Journal of advanced research

Article Title: Metformin attenuates colitis via blocking STAT3 acetylation by reducing acetyl-CoA production.

doi: 10.1016/j.jare.2025.03.058

Figure Lengend Snippet: Fig. 4. Metformin attenuates colitis by suppressing the STAT3 signaling pathway. (A) Experimental flow chart: STAT3fl/fl and STAT3DIEC mice (n = 6) were administered metformin (200 mg/kg) or PBS intraperitoneally, followed by continuous delivery of 3 % DSS in their drinking water for 7 days. (B-C) Changes in body weight and DAI of mice were monitored from day 1. (D-E) A comparison of colon length and morphology in mice. (F) Representative images of H&E morphology, along with IHC analysis of AB-PAS and MUC-2. (G) Histological scores of colon tissue from mice in various treatment groups. (H-I) Representative immunofluorescence images of ZO-1 and TUNEL staining. (J) Scanning electron microscopy images of mouse colonic tissue sections. (K) WB analysis of colonic protein expression in mice, including STAT3, p-STAT3Y705 , E-cadherin, Occludin, Bcl-2, and Bax. (L) IHC analysis of p-STAT3Y705 expression in mouse colonic tissue. (M) WB analysis of protein expression in NCM460 cells. The cells were transfected with siSTAT3 or siNC using Lipofectamine 3000, followed by treatment with or without metformin (200 lM for 24 h) and stimulation with LPS (1 lg/ml for 12 h). (N-P) Representative IF images showing ZO-1, p-STAT3Y705 , and STAT3 expression in cells. (Q) WB analysis of relevant protein levels in NCM460 cells. The cells were cocultured with LPS (1 lg/mL for 12 h) and transfected with a STAT3 overexpression plasmid (STAT3WT ) or negative control (NC), with or without metformin pretreatment (200 lM for 24 h). (R) WB assessment of relevant protein levels in NCM460 cells. The cells were treated with DMSO or Colivelin TFA (25 lg/ml for 13 h, Med Chem Express, #HY-P1061A) following treatment with LPS (1 lg/ml for 12 h) and metformin (200 lM for 24 h).

Article Snippet: The following primary antibodies were used: Bax (Proteintech, #50599–2-Ig), STAT3 (Cell Signaling Technology, #124H6), pSTAT3 (Cell Signaling Technology, #9145S), ac-STAT3 (Cell Signaling Technology, #2523), ZO-1 (Proteintech, #21773–1-AP), Lamin B (Proteintech, #12987–1-AP), b-actin (Proteintech, #66009–1- Ig), Occludin (Proteintech, #27260–1-AP), Bcl-2 (Abclonal, #19693; AbMART, #T40056), E-cadherin (Proteintech, #20874–1- AP), Histone H3 (Proteintech, #17168–1-AP), HA tag (Proteintech, #51064–2-AP), acetyl-Histone H3-K9 (AbMART, #P37961-16), caspase-3 (Abclonal, #19654).

Techniques: Comparison, TUNEL Assay, Staining, Electron Microscopy, Expressing, Paraffin-embedded Immunohistochemistry, Transfection, Over Expression, Plasmid Preparation, Negative Control

Journal: Journal of advanced research

Article Title: Metformin attenuates colitis via blocking STAT3 acetylation by reducing acetyl-CoA production.

doi: 10.1016/j.jare.2025.03.058

Figure Lengend Snippet: Fig. 5. Metformin supresses STAT3 acetylation to alleviate intestinal inflammation. (A-B) WB and IF analysis of protein expression in NCM460 cells treated with Ex527 (50 lM for 24 h) or DMSO, followed by LPS stimulation. (C-D) WB and IF analysis of protein expression in NCM460 cells treated with metformin (200 lM for 24 h) or SRT1720 (5 lM for 12 h, Med Chem Express, #HY-10532), followed by LPS stimulation. (E) Schematic of experimental design. STAT3fl/fl and STAT3DIEC mice (n = 5) were intraperitoneally administered Ex527 (10 mg/kg, 7 days) or DMSO, followed by metformin (200 mg/kg) and 3 % DSS treatment for 7 days. (F) Body weight changes and DAI scores. (G) Colon length and macroscopic morphology. (H) Representative H&E-stained colon sections and IHC analysis of AB-PAS and MUC-2. (I) Histopathological scoring of H&E-stained colon sections. (J) Statistical analysis of Western blot results. STAT3fl/fl and STAT3DIEC mice (n = 5) were intraperitoneally administered Ex527 (10 mg/kg, 7 days) or DMSO, followed by metformin (200 mg/kg) and 3 % DSS treatment for 7 days. (K) Statistical analysis of Western blot results. STAT3WT - or STAT3K685 -transfected NCM460 cells were treated with Ex527 (50 lM) or DMSO for 24 h, followed by LPS + Met. (L) Scanning electron microscopy (SEM) images of colonic epithelium. (M) WB analysis of colonic protein expression in mice. (N) IHC staining of p-STAT3Y705 in STAT3fl/fl mouse colons. (O) IF staining of ac-STAT3K685 in STAT3fl/fl mouse colons. (P-R) IF images of ZO- 1, p-STAT3Y705 , and ac-STAT3K685 in NCM460 cells treated with Ex527 (50 lM) or DMSO for 24 h, followed by LPS + Met. (S) WB analysis of protein levels in STAT3WT - or STAT3K685 -transfected NCM460 cells treated with Ex527 (50 lM) or DMSO for 24 h, followed by LPS + Met.

Article Snippet: The following primary antibodies were used: Bax (Proteintech, #50599–2-Ig), STAT3 (Cell Signaling Technology, #124H6), pSTAT3 (Cell Signaling Technology, #9145S), ac-STAT3 (Cell Signaling Technology, #2523), ZO-1 (Proteintech, #21773–1-AP), Lamin B (Proteintech, #12987–1-AP), b-actin (Proteintech, #66009–1- Ig), Occludin (Proteintech, #27260–1-AP), Bcl-2 (Abclonal, #19693; AbMART, #T40056), E-cadherin (Proteintech, #20874–1- AP), Histone H3 (Proteintech, #17168–1-AP), HA tag (Proteintech, #51064–2-AP), acetyl-Histone H3-K9 (AbMART, #P37961-16), caspase-3 (Abclonal, #19654).

Techniques: Expressing, Staining, Western Blot, Transfection, Electron Microscopy, Immunohistochemistry

Journal: Journal of advanced research

Article Title: Metformin attenuates colitis via blocking STAT3 acetylation by reducing acetyl-CoA production.

doi: 10.1016/j.jare.2025.03.058

Figure Lengend Snippet: Fig. 6. Elevated acetyl-CoA levels exacerbate UC by promoting STAT3 acetylation. (A) Schematic representation of the experimental model. WT mice (n = 6) were gavaged with acetate (500 mg/kg) or PBS for 3 weeks, followed by treatment with metformin (200 mg/kg) and 3 % DSS for 7 days. (B) Relative acetyl-CoA levels in mice. (C-D) Representative IF images of ac-STAT3K685 and IHC images of p-STAT3Y705 in mice. (E) Alterations in body weight and DAI. (F-G) Gross morphology and colon length measurements in mice. (H) Representative images of H&E staining for morphological analysis and IHC analysis of AB-PAS and MUC-2 expression. (I) Histological evaluation of colonic tissue based on morphological examination of colon sections. (J-K) Representative immunofluorescence images of ZO-1 and TUNEL staining, along with quantitative analysis. (L) Representative electron microscopy images of colonic tissue segments. (M) WB analysis of colonic protein expression in mice. (N) ELISA analysis of relative intracellular acetyl-CoA levels (n = 3). NCM460 cells were exposed to acetate (5 mM) or PBS for 24 h, followed by treatment with metformin and LPS. (O) WB analysis of relevant protein expression in cells treated with acetate or PBS. (P-R) Representative immunofluorescence images of ZO-1, p-STAT3Y705 , and ac-STAT3K685 in cells.

Article Snippet: The following primary antibodies were used: Bax (Proteintech, #50599–2-Ig), STAT3 (Cell Signaling Technology, #124H6), pSTAT3 (Cell Signaling Technology, #9145S), ac-STAT3 (Cell Signaling Technology, #2523), ZO-1 (Proteintech, #21773–1-AP), Lamin B (Proteintech, #12987–1-AP), b-actin (Proteintech, #66009–1- Ig), Occludin (Proteintech, #27260–1-AP), Bcl-2 (Abclonal, #19693; AbMART, #T40056), E-cadherin (Proteintech, #20874–1- AP), Histone H3 (Proteintech, #17168–1-AP), HA tag (Proteintech, #51064–2-AP), acetyl-Histone H3-K9 (AbMART, #P37961-16), caspase-3 (Abclonal, #19654).

Techniques: Staining, Expressing, TUNEL Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Exosome-derived circ-001422 promotes tumor-associated macrophage M2 polarization to accelerate the progression of glioma

doi: 10.1038/s42003-024-07134-0

Figure Lengend Snippet: A Three online tool including PROMO, ChipBase and Human TFDB was used to analyzed the transcription factor of WHSC1 (host gene of circ-001422). B Chip-qPCR was used to analyzed the binding between potential transcription factor and WHSC1. Data were expressed as mean ± SD for triplicate experiments. C Motif of STAT3 was obtained from JASPAR. D Potential binding sites between STAT3 and WHSC1 were predicted by JASPAR. E Mutation of binding site 4 significantly reduced the binding with STAT3 and WHSC1. Data were expressed as mean ± SD for triplicate experiments. F Chip-qPCR indicated that STAT3 significantly bind with the binding site 4 of WHSC1. Data were expressed as mean ± SD for triplicate experiments. G Design specific primers for amplifying pre-mRNA of WHSC1, WHSC1 mRNA and circ-001422. H STAT3 elevated the levels of pre-mRNA of WHSC1, while knockdown of STAT3 reduced the levels. Data were expressed as mean ± SD for triplicate experiments. I STAT3 reduced the levels of mRNA of WHSC1, while knockdown of STAT3 increased the mRNA levels of WHSC1. Data were expressed as mean ± SD for triplicate experiments. J STAT3 elevated the levels of circ-001422, while knockdown of STAT3 reduced the levels. Data were expressed as mean ± SD for triplicate experiments. * P < 0.05; ** P < 0.01.

Article Snippet: The proteins were then transferred onto PVDF membranes with a pore diameter of 0.45 μm (Millipore, USA), blocked in 5% BSA for 30 min, and incubated with primary antibodies including TGF-β1 (1:1000, Cat No : 21898-1-AP, Proteintech), ARG1 (1:5000, Cat no. 16001-1-AP, Proteintech), IL-10 (1:2000, Cat no. 60269-1-Ig), HSP70 (1:5000, Cat no. 10995-1-AP, Proteintech), CD81 (1:1000, Cat no. 66866-1-Ig, Proteintech), CD9 (1:1000, Cat no : 20597-1-AP, Proteintech), TSG101 (1:2000, Cat no. 28283-1-AP, Proteintech), ace-STAT3 (1:1000, Cat no. #2523, CST), p-STAT3 (Y705) (1:2000, Cat no. #9145, CST), STAT3 (1:2000, Cat No : 10253-2-AP, Proteintech), NF-κB-p65 (1:5000, Cat no. 80979-1-RR, Proteintech), p300 (1:500, Cat no. 20695-1-AP, Proteintech), NF-κB-p65 (S536) (1:1000, Cat no. #3033, CST), and GAPDH (1:50000, Cat no. 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: ChIP-qPCR, Binding Assay, Mutagenesis, Knockdown

Journal: Communications Biology

Article Title: Exosome-derived circ-001422 promotes tumor-associated macrophage M2 polarization to accelerate the progression of glioma

doi: 10.1038/s42003-024-07134-0

Figure Lengend Snippet: A Protein profile analysis of circ-001422 binding protein, especially for the p300 and STAT3 in the THP-1 cells. B RIP assays indicated that p300 and STAT3 bind with circ-001422. C Circ-001422 was co-located with p300 and STAT3 in nuclear of THP-1 cells. Data were expressed as mean ± SD for triplicate experiments. D Western blot and immunoprecipitation experiments demonstrate that circ-001422 overexpression in THP-1 cells enhances p300 and STAT3 acetylation, leading to STAT3/NF- κB nuclear translocation. Knockdown of circ-001422 in THP-1 cells induced the opposite effects. E Immunofluorescence analysis shows reduced expression of circ-001422 decreased the co-localization of STAT3, and p300 in macrophage like THP-1 cells. F Luciferase reporter assay confirmed the impact of circ-001422 on the transcriptional activity of STAT3 and NF-κB in the THP-1 cells. Data were expressed as mean ± SD for triplicate experiments. * P < 0.05; ** P < 0.01.

Article Snippet: The proteins were then transferred onto PVDF membranes with a pore diameter of 0.45 μm (Millipore, USA), blocked in 5% BSA for 30 min, and incubated with primary antibodies including TGF-β1 (1:1000, Cat No : 21898-1-AP, Proteintech), ARG1 (1:5000, Cat no. 16001-1-AP, Proteintech), IL-10 (1:2000, Cat no. 60269-1-Ig), HSP70 (1:5000, Cat no. 10995-1-AP, Proteintech), CD81 (1:1000, Cat no. 66866-1-Ig, Proteintech), CD9 (1:1000, Cat no : 20597-1-AP, Proteintech), TSG101 (1:2000, Cat no. 28283-1-AP, Proteintech), ace-STAT3 (1:1000, Cat no. #2523, CST), p-STAT3 (Y705) (1:2000, Cat no. #9145, CST), STAT3 (1:2000, Cat No : 10253-2-AP, Proteintech), NF-κB-p65 (1:5000, Cat no. 80979-1-RR, Proteintech), p300 (1:500, Cat no. 20695-1-AP, Proteintech), NF-κB-p65 (S536) (1:1000, Cat no. #3033, CST), and GAPDH (1:50000, Cat no. 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Over Expression, Translocation Assay, Knockdown, Immunofluorescence, Expressing, Luciferase, Reporter Assay, Activity Assay

Journal: Communications Biology

Article Title: Exosome-derived circ-001422 promotes tumor-associated macrophage M2 polarization to accelerate the progression of glioma

doi: 10.1038/s42003-024-07134-0

Figure Lengend Snippet: A Western blotting experiments demonstrated that circ-001422 activated STAT3/NF-κB-dependent STAT3 acetylation modification. B Western blotting indicated that exosomal circ-001422 was shown to activate STAT3/NF-κB-dependent STAT3 acetylation. C Immunofluorescence assays revealed that exosome-derived circ-001422 modulated STAT3 through acetylation and nuclear translocation of NF-κB. D Double fluorescein reporter experiments confirmed that exosome-derived circ-001422 regulated the transcriptional activity of STAT3 and NF-κB via STAT3 acetylation. Data were expressed as mean ± SD for triplicate experiments. E Flow cytometry results indicated that exosome-derived circ-001422 increased the positive rate of CD206 in THP-1 cells via STAT3 acetylation. Data were expressed as mean ± SD for triplicate experiments. F qRT-PCR results indicated that exosome-derived circ-001422 increased M2 biomarkers and reduced M1 biomarkers in THP-1 cells via STAT3 acetylation. * P < 0.05; ** P < 0.01. (1) NC. (2) sh-#1 U87 Exo; (3) sh-#1 U87 Exo + STAT3-K685R; (4) sh-#1 U87 Exo + STAT3-K685Q; (5) sh-#1 U87 Exo + STAT3-WT.

Article Snippet: The proteins were then transferred onto PVDF membranes with a pore diameter of 0.45 μm (Millipore, USA), blocked in 5% BSA for 30 min, and incubated with primary antibodies including TGF-β1 (1:1000, Cat No : 21898-1-AP, Proteintech), ARG1 (1:5000, Cat no. 16001-1-AP, Proteintech), IL-10 (1:2000, Cat no. 60269-1-Ig), HSP70 (1:5000, Cat no. 10995-1-AP, Proteintech), CD81 (1:1000, Cat no. 66866-1-Ig, Proteintech), CD9 (1:1000, Cat no : 20597-1-AP, Proteintech), TSG101 (1:2000, Cat no. 28283-1-AP, Proteintech), ace-STAT3 (1:1000, Cat no. #2523, CST), p-STAT3 (Y705) (1:2000, Cat no. #9145, CST), STAT3 (1:2000, Cat No : 10253-2-AP, Proteintech), NF-κB-p65 (1:5000, Cat no. 80979-1-RR, Proteintech), p300 (1:500, Cat no. 20695-1-AP, Proteintech), NF-κB-p65 (S536) (1:1000, Cat no. #3033, CST), and GAPDH (1:50000, Cat no. 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Western Blot, Modification, Immunofluorescence, Derivative Assay, Translocation Assay, Activity Assay, Flow Cytometry, Quantitative RT-PCR