ac stat3 cell signaling  (Cell Signaling Technology Inc)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A-485 alleviates fibrosis and apoptosis in kidney by disrupting tandem activation of acetylation and phosphorylation on STAT3.

doi: 10.1016/j.biopha.2025.118217

Figure Lengend Snippet: Fig. 7. Effects of A-485 on acetylation and phosphorylation of STAT3 in UUO kidney. (A) The representative western blot images and quantification of p-STAT3 (Y705), p-STAT3(S727), ac-STAT3(K685) and total STAT3 in Sham and UUO mice (n = 6). (B) The representative western blot images and quantification of p-STAT3 (Y705), p-STAT3 (S727), ac-STAT3 (K685) and total STAT3 in UUO mice with or without A-485 treatment (n = 6). (C-D) The quantification of p-STAT3(Y705) and ac-STAT3 (K685) expression in UUO mice detected by immunohistochemical staining (n = 6, scale bar = 50μm). (E) The representative western blot images and quantification of p300 and CBP in Sham and UUO mice (n = 6). (F) The representative western blot images and quantification of p300 and CBP in UUO mice with or without A-485 treatment (n = 6). (G) The p300 catalytic activity in mice kidney (n = 6). Values are expressed as means ± SEM. *p < 0.05, * *p < 0.01. Sham, sham operation; UUO, unilateral ureteral obstruction. CBP, CREB binding protein.

Article Snippet: The antibodies used in the present study are listed as below: antibodies against Bcl-2 (ab196495), Collagen I (ab34710); antibodies against STAT3 (10253–2-AP), Bax (50599–2-Ig), α-SMA (14395–1-AP), Fibronectin (15613–1-AP), E-Cadherin (20874–1-AP) and GAPDH (10494–1-AP) (Proteintech, Chicago, IL, USA); antibodies against Acetyl-STAT3 (Lys685) (#2523), Cleaved Caspase-3 (#9661), p300 (#67621), CBP(#74384SF), Acetylated-Lysine (#9441), PhosphoSTAT3 (S727) (#49081) and Phospho-STAT3 (Y705) (#9145) (Cell Signaling Technology, Beverly, MA, USA); antibodies against NDUFS1 (ER1803–08) and SDHB (ER1803–63) (Hua’an Biotechnology, Hangzhou, China); antibody against COX4 (sc-517553) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China); Inhibitor A-485 (1889279–16–6) (GlpBio, Montclair, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Immunohistochemical staining, Staining, Activity Assay, Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A-485 alleviates fibrosis and apoptosis in kidney by disrupting tandem activation of acetylation and phosphorylation on STAT3.

doi: 10.1016/j.biopha.2025.118217

Figure Lengend Snippet: Fig. 8. Effects of A-485 on acetylation and phosphorylation of STAT3 in TGF-β1-induced HK-2 cells. (A) The representative western blot images and quantification of p-STAT3(Y705), p-STAT3(S727), ac-STAT3 (K685) and STAT3 in HK-2 cells treated with different A-485 concentrations (n = 3). (B) The immunofluorescence staining of p-STAT3(Y705) and ac-STAT3 (K685) in HK-2 cells (scale bar = 50μm). (C) The representative western blot images and quantification of p300 and CBP in HK-2 cells treated with different A-485 concentrations (n = 3). (D) The p300 catalytic activity in HK-2 cells (n = 5). Values are expressed as means ± SEM. *p < 0.05, * *p < 0.01. CBP, CREB binding protein.

Article Snippet: The antibodies used in the present study are listed as below: antibodies against Bcl-2 (ab196495), Collagen I (ab34710); antibodies against STAT3 (10253–2-AP), Bax (50599–2-Ig), α-SMA (14395–1-AP), Fibronectin (15613–1-AP), E-Cadherin (20874–1-AP) and GAPDH (10494–1-AP) (Proteintech, Chicago, IL, USA); antibodies against Acetyl-STAT3 (Lys685) (#2523), Cleaved Caspase-3 (#9661), p300 (#67621), CBP(#74384SF), Acetylated-Lysine (#9441), PhosphoSTAT3 (S727) (#49081) and Phospho-STAT3 (Y705) (#9145) (Cell Signaling Technology, Beverly, MA, USA); antibodies against NDUFS1 (ER1803–08) and SDHB (ER1803–63) (Hua’an Biotechnology, Hangzhou, China); antibody against COX4 (sc-517553) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China); Inhibitor A-485 (1889279–16–6) (GlpBio, Montclair, USA).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Staining, Activity Assay, Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A-485 alleviates fibrosis and apoptosis in kidney by disrupting tandem activation of acetylation and phosphorylation on STAT3.

doi: 10.1016/j.biopha.2025.118217

Figure Lengend Snippet: Fig. 9. The relationship between phosphorylation and acetylation of STAT3 in TGF-β1 stimulated HK-2 cells. (A) The representative western blot images and quantification of p-STAT3 (Y705), ac-STAT3 (K685) and STAT3 in HK-2 cells stimulated with different TGF-β1 concentrations (n = 3). (B) The representative western blot images and quantification of p-STAT3 (Y705), ac-STAT3 (K685) and STAT3 in HK-2 cells with the transfection of the plasmids of STAT3 WT, K685R, K685Q (n = 3). (C) The representative western blot images and quantification of p-STAT3 (Y705), ac-STAT3 (K685) and STAT3 in HK-2 cells with the transfection of the plasmids of STAT3 WT, Y705F, Y705D (n = 3). (D) The immunofluorescence staining of p-STAT3(Y705), ac-STAT3 (K685), α-SMA in HK-2 cells (scale bar = 50μm). Values are expressed as means ± SEM. * *p < 0.01. KO, knockout; WT, wild type.

Article Snippet: The antibodies used in the present study are listed as below: antibodies against Bcl-2 (ab196495), Collagen I (ab34710); antibodies against STAT3 (10253–2-AP), Bax (50599–2-Ig), α-SMA (14395–1-AP), Fibronectin (15613–1-AP), E-Cadherin (20874–1-AP) and GAPDH (10494–1-AP) (Proteintech, Chicago, IL, USA); antibodies against Acetyl-STAT3 (Lys685) (#2523), Cleaved Caspase-3 (#9661), p300 (#67621), CBP(#74384SF), Acetylated-Lysine (#9441), PhosphoSTAT3 (S727) (#49081) and Phospho-STAT3 (Y705) (#9145) (Cell Signaling Technology, Beverly, MA, USA); antibodies against NDUFS1 (ER1803–08) and SDHB (ER1803–63) (Hua’an Biotechnology, Hangzhou, China); antibody against COX4 (sc-517553) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China); Inhibitor A-485 (1889279–16–6) (GlpBio, Montclair, USA).

Techniques: Phospho-proteomics, Western Blot, Transfection, Immunofluorescence, Staining, Knock-Out

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