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: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 6. ASFV promotes IL-6 expression to activate STAT3 phosphorylation by C315R. (A) RT-PCR analysis of IL-6 mRNA expression in 3D4/21 cells infected with ASFV of different MOIs at different time points post infection, calculated using the 2−∆∆CT method. The data were analyzed using GraphPad Prism 8.0.2 software. The data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01; **** p < 0.0001 determined by two-tailed Student’s t-test. ns, no significance). (B) Immunoblotting of whole cell lysates from 3D4/21 cells infected with ASFV at different time points and MOIs, probing for pSTAT3 (Ser727), STAT3, IL-6, p30, p72, and the cytosolic markers α-β-tubulin. (C) Confocal microscopy of 3D4/21 cells infected with ASFV at 24 hpi, showing colocalization of p30 (red) and pSTAT3 (Ser727) (green). Nuclei were counterstained with DAPI (blue). Scale bar: 25 µm. (D) Fluorescence intensity analysis of individual cells in C. (E) RT-PCR analysis of IL-6 expression in 3D4/21 cells transfected with either the empty vector or pcDNA3.1-HA-C315R, calculated by the 2−∆∆CT method. (F) Immunoblotting of lysates from 3D4/21 cells transfected with pcDNA3.1-HA-C315R, probing for pSTAT3 (Ser727), STAT3, IL-6, HA, and α-β-tubulin. (G) Confocal microscopy showing colocalization

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Expressing, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Infection, Software, Two Tailed Test, Western Blot, Confocal Microscopy, Fluorescence, Transfection, Plasmid Preparation

Journal: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 7. Inhibition of STAT3 phosphorylation reduces ASFV replication and DNA synthesis. (A) Cy- totoxicity of Stattic was assessed using the CCK8 assay, which was represented as the average of three independent experiments. The data were analyzed using GraphPad Prism 8.0.2 software. (B) 3D4/21 cells were pretreated with Stattic or DMSO (control) for 4 h, followed by ASFV infection. IL-6 and p30 mRNA levels were analyzed by RT-PCR and calculated using the 2−∆∆CT method. Data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01 determined by two-tailed Student’s t-test. ns, no significance). (C) Immunoblotting of 3D4/21 cell lysates treated with Stattic or DMSO for 4 h, probing for pSTAT3 (Ser727), STAT3, p30, and the cytosolic marker β-Tubulin. (D) PAMs pretreated with Stattic and infected with ASFV showed a significant reduction in viral yields from 5.37 to 2.00 HAD50/mL. The data were analyzed using GraphPad Prism 8.0.2 software. (E) 3D4/21 cells pretreated with Stattic or DMSO were infected with GFP-ASFV (green) for 24 h. Fluorescence images were captured using a fluorescence microscope (scale bar: 300 µm). (F) 3D4/21 cells, mock-infected, ASFV-infected, or ASFV-infected with Stattic for 12 h, were pulsed with 10 µM EdU for 2 h. DNA synthesis was measured by flow cytometry using a 488 nm laser for EdU detection. (G) The data from (F) were analyzed using GraphPad Prism 8.0.2 software.

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Inhibition, Phospho-proteomics, DNA Synthesis, CCK-8 Assay, Software, Control, Infection, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Western Blot, Marker, Fluorescence, Microscopy, Flow Cytometry

Journal: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 8. Model of C315R-mediated activation of the IL-6 STAT3 signaling axis facilitating ASFV infection. C315R promotes the transcription of the pro-inflammatory cytokine IL-6, which in turn activates STAT3 phosphorylation (pSTAT3). Phosphorylated STAT3 translocate to the nucleus, where it enhances the transcription of viral genes, thereby supporting ASFV replication.

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Activation Assay, Infection, Phospho-proteomics

Journal: Scientific reports

Article Title: Starvation-induced HBP metabolic reprogramming and STAM2 O-GlcNAcylation facilitate bladder cancer metastasis.

doi: 10.1038/s41598-025-92579-4

Figure Lengend Snippet: Fig. 5. STAM2 S375 O-GlcNAcylation facilitates the JAK2–STAT3 signaling pathway. (A) STAM2 protein levels were measured using western blotting. (B) JAK2/p-JAK2 and STAT3/p-STAT3 protein levels were measured using western blotting in NC cells, STAM2-overexpressing cells and STAM2-knockout cells. (C) Quantitative western blot analysis of B. (D) JAK2/p-JAK2 and STAT3/p-STAT3 protein levels were measured using western blot in UM-UC-3 cells transfected with STAM2-HA or STAM2 S375-HA and treated with PUGNAc or not. (E) Quantitative western blot analysis of D. (F-G) Proliferation, migration, and invasion were measured using CCK-8 and Transwell assays in NC cells or cells transfected with STAM2-HA or STAM2 S375-HA after treatment with JSI-124. (H) EMT marker protein levels were measured using western blotting. (I) Quantitative western blot analysis of H. All PVDF bands were transferred and tested in the same western blot experiments. All the results represent three distinct repetitions. Statistical significance is denoted as ***p < 0.001, **p < 0.01, and *p < 0.05, indicating significant differences between two groups.

Article Snippet: The antibodies used in the study were as follows: STAM2 (Proteintech, 13009-1-AP), HA (Proteintech,51064-2-AP), E-cadherin (Proteintech, 20874-1- AP), N-cadherin (Proteintech, 22018-1-AP), Vimentin (Proteintech, 10366-1-AP), JAK2 (CST, #3230), p-JAK2 (CST, #3771), stat3 (CST, #12640), p-stat3 (CST, #94994), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Western Blot, Knock-Out, Transfection, Migration, CCK-8 Assay, Marker

Journal: Scientific reports

Article Title: Starvation-induced HBP metabolic reprogramming and STAM2 O-GlcNAcylation facilitate bladder cancer metastasis.

doi: 10.1038/s41598-025-92579-4

Figure Lengend Snippet: Fig. 6. STAM2 S375 O-GlcNAcylation promotes tumor proliferation and metastasis in vivo. (A) Schematic representation of the establishment of UM-UC-3-derived xenografts. (B) Representative images of xenograft tumors 21 days after subcutaneous injection. C Tumor volumes were measured every 3 days. (D) Weights of xenograft tumors 21 days after subcutaneous injection. (E-H) Protein levels of EMT marker JAK2/p-JAK2 and STAT3/p-STAT3 were measured using western blotting. All PVDF bands were transferred and tested in the same western blot experiments, and the experiments performed in triplicate. (I-L) Bioluminescence detection and bioluminescence quantitative analysis of the lung (I-J) and popliteal lymph nodes (K-L). (M- N) Representative image of the popliteal lymphatic metastasis model, along with quantitative analysis of the volume of popliteal lymph nodes.

Article Snippet: The antibodies used in the study were as follows: STAM2 (Proteintech, 13009-1-AP), HA (Proteintech,51064-2-AP), E-cadherin (Proteintech, 20874-1- AP), N-cadherin (Proteintech, 22018-1-AP), Vimentin (Proteintech, 10366-1-AP), JAK2 (CST, #3230), p-JAK2 (CST, #3771), stat3 (CST, #12640), p-stat3 (CST, #94994), and β-actin (Proteintech, 66009-1-Ig).

Techniques: In Vivo, Derivative Assay, Injection, Marker, Western Blot

Journal: iScience

Article Title: RGS2 is an innate immune checkpoint for suppressing Gαq-mediated IFNγ generation and lung injury

doi: 10.1016/j.isci.2025.111878

Figure Lengend Snippet:

Article Snippet: Rabbit Monoclonal anti-phospho-STAT3 (S727) , Cell Signaling , Cat # 9134S.

Techniques: Recombinant, Protease Inhibitor, Red Blood Cell Lysis, Construct, Control, Software