Journal: Cell Reports Medicine

Article Title: A patient-derived T cell lymphoma biorepository uncovers pathogenetic mechanisms and host-related therapeutic vulnerabilities

doi: 10.1016/j.xcrm.2025.102029

Figure Lengend Snippet:

Article Snippet: anti-pSTAT3 (clone M9C6) , Cell Signaling Technology , 4113; RRID: AB_2198588.

Techniques: Transduction, Recombinant, Saline, Software, Sterility, Transferring, Ointment

Journal: BMC Developmental Biology

Article Title: Interleukin-6 promotes primitive endoderm development in bovine blastocysts

doi: 10.1186/s12861-020-00235-z

Figure Lengend Snippet: IL6 preferentially stimulates pSTAT3 Y705 nuclear localization in PE cells. Day 9 blastocysts were treated with 100 ng/ml IL6 for 30 min before fixation and fluorescent immunostaining. Panel a : Example of a blastocyst immunostained for pSTAT3 Y705 (red) and NANOG (blue). Dual-positive nuclei will be purple. Panel b : Number and percentage of blastocysts with either some or all ICM nuclei staining for both NANOG and pSTAT3 Y705 ( n = 24 pSTAT3 Y705 positive blastocysts over 4 replicate studies) Panel c : Example of a blastocyst immunostained for pSTAT3 Y705 (green) and GATA6 (red). Dual-positive nuclei will be yellow. Panel d : Number and percentage of blastocysts with either some or all ICM nuclei staining for both GATA6 and pSTAT3 Y705 ( n = 26 pSTAT3 Y705 positive blastocysts over 3 replicate studies)

Article Snippet: Blastocysts were then incubated for 1 h at room temperature or overnight at 4 °C with either rabbit anti-pSTAT3 Y705 (Cell Signaling Technologies; 9145 T; 1:100) and mouse anti-NANOG (same as above; 1:200), or with mouse anti-pSTAT3 Y705 (Santa Cruz Biotechnology, Dallas, TX; sc-8059; 1:200) and rabbit anti-GATA6 (same as above; 1:500).

Techniques: Immunostaining, Staining

Journal: Oncogene

Article Title: The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

doi: 10.1038/sj.onc.1207842

Figure Lengend Snippet: Figure 6 Variable response of NF-kB to UCN-01 treatment. The transcriptional activity of NF-kB is suppressed by UCN-01 treatment (1 mM, 48 h) in SW48 cell line, but it is activated in WiDr, HT-29 and LS513 cells Figure 7 UCN-01 treatment can inhibit both Ser727 and Tyr705 phosphorylation of STAT3 a. (a) Constitutive STAT3 phosphor- ylation at Ser727 is detectable in all cell lines. It is inhibited by UCN-01 (1 mM, 48 h) only in the apoptosis-performing cell lines. (b) STAT3 phosphorylation at Tyr705 is not detectable in any of the cell lines (shown LS513). It is inducible by IFNa and is inhibited within 1.5 h by UCN-01 treatment

Article Snippet: The following antibodies were employed: rabbit anti-BAX antibody (N20), goat anti-Akt1 antibody (C-20), mouse monoclonal anti-phosphotyrosine STAT3 antibody (clone B-7) (all from Santa Cruz Biotechnology, Heidelberg, Germany); mouse monoclonal anti-PARP antibody (clone C2-10), and mouse monoclonal anti-Bcl-xL (clone 2H12) (all from BD Pharmingen, Heidelberg, Germany); mouse monoclonal anti-b-actin antibody (clone AC-15) (Sigma-Aldrich Chemie, Taufkirchen, Germany).

Techniques: Activity Assay, Phospho-proteomics

Journal: Oncogene

Article Title: The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

doi: 10.1038/sj.onc.1207842

Figure Lengend Snippet: Figure 8 Activation of STAT3 does not lead to Bcl-xL transcrip- tion. (a) Exogenous STAT3 is phosphorylated at Tyr705 after stimulation of SW48 cells with IL-6. (b) IL-6 stimulation activates the transcriptional activity of STAT3 as demonstrated by the increased activity of the luciferase reporter. (c) Bcl-xL transcription is not affected by the transcriptionally active STAT3. Total RNA was isolated from the same cells as used in (b), and the semiquantitative RT–PCR was carried out with the cycle number in the linear range of the amplification. b-actin was amplified for the control of the amounts of mRNA used in each lane

Article Snippet: The following antibodies were employed: rabbit anti-BAX antibody (N20), goat anti-Akt1 antibody (C-20), mouse monoclonal anti-phosphotyrosine STAT3 antibody (clone B-7) (all from Santa Cruz Biotechnology, Heidelberg, Germany); mouse monoclonal anti-PARP antibody (clone C2-10), and mouse monoclonal anti-Bcl-xL (clone 2H12) (all from BD Pharmingen, Heidelberg, Germany); mouse monoclonal anti-b-actin antibody (clone AC-15) (Sigma-Aldrich Chemie, Taufkirchen, Germany).

Techniques: Activation Assay, Activity Assay, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: We further examined whether ODN priming and/or later IL-15 exposure influences STAT1 and STAT3 tyrosine phosphorylation at residues, Y701 and Y705 respectively. Intracellular levels of (A) pSTAT1Y701 and (B) pSTAT3Y705, as well as (C) pSTAT5Y694 were monitored by immunofluorescence staining and flow cytometry of viable-gated cells in a new cohort of 6 B-CLL clones. Expression was assessed in both ODN-primed or unprimed cultures ± subsequent 60 min exposure to IL-15. Statistical analyses were performed with a paired, 2-sided t-test when pooled data passed normality tests. In cases of non-parametric data, the Wilcoxon Signed Rank test was employed. The latter applied to pSTAT1: untreated vs ODN only; ODN only versus ODN + IL-15, and pSTAT3: untreated vs IL-15 only.

Article Snippet: For brevity, we will refer to positive binding as representing pSTAT5 Y694 . (c) PE-mouse anti-pSTAT3 Y705 mAb or PE-isotype control (BD Biosciences) and (d) AF-488 mouse anti-pSTAT1 Y701 or AF-488 isotype control (BD Biosciences).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Clone Assay, Expressing

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