phosphorylated ser727  (Cell Signaling Technology Inc)

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Impact of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway on tumor stem cell self-renewal and migration invasion. (A, B) Western blot analysis of JAK-STAT and nuclear factor kappa B (NF-κB) signaling pathway-related proteins: protein expression levels of phosphorylated-Janus kinase 2 (p-JAK2), JAK2, phosphorylated signal transducer and activator of transcription (p-STAT3), STAT3, NF-κB, and phosphorylated NF-κB (p-NF-κB) in FTC238-S cells co-cultured with different modified myeloid‑derived suppressor cells (MDSCs) (A), and protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in FTC238-S cells treated with anti-immunoglobulin G (anti-IgG) or anti-C-X-C motif chemokine ligand 8 (anti-CXCL8) antibodies (B). (C) Schematic diagram of monocyte treatment and co-culturing with FTC238-S cells. (D) Western blot analysis of JAK-STAT signaling pathway-related proteins in FTC238-S cells from each group. (E) Protein expression levels of stemness markers neuroepithelial stem cell protein (NESTIN), octamer-binding transcription factor 4 (OCT4), and SRY-box transcription factor 2 (SOX2) in FTC238-S cells from each group examined by Western blot. (F) Cell sphere formation assay assessing the sphere formation capability of FTC238-S cells in co-culture systems of each group. (G) Clonogenic assay evaluating the clonogenic capacity of FTC238-S cells in co-culture systems of each group. (H) Cell Counting Kit-8 (CCK-8) assay measuring the proliferation ability of FTC238-S cells in co-culture systems of each group. (I) Transwell assay determining the migration and invasion capability of FTC238-S cells in co-culture systems of each group, ∗ P < 0.05 compared between the two groups, and all cell experiments were repeated three times. M_oe-NC + S_sh-NC: FTC238-S cells transfected with short hairpin RNA-negative control (sh-NC) and treated with conditioned medium from monocytes transfected with oe-NC; M_oe-CXCL8 + S_sh-NC: FTC238-S cells transfected with sh-NC and treated with conditioned medium from monocytes transfected with oe-CXCL8; M_oe-CXCL8 + S_sh-SDC1: FTC238-S cells transfected with short hairpin RNA-SDC1 (sh-SDC1) and treated with conditioned medium from monocytes transfected with oe-CXCL8; M_oe-CXCL8 + S_DMSO: FTC238-S cells treated with conditioned medium from monocytes transfected with oe-CXCL8 and supplemented with an equal amount of dimethyl sulfoxide (DMSO); M_oe-CXCL8 + S_SD_1008: FTC238-S cells treated with conditioned medium from monocytes transfected with oe-CXCL8 and supplemented with SD-1008; THP-1 cells: Tohoku Hospital Pediatrics-1 cells; OD: optical density.

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: Migration, Western Blot, Expressing, Cell Culture, Modification, Binding Assay, Tube Formation Assay, Co-Culture Assay, Clonogenic Assay, Cell Counting, CCK-8 Assay, Transwell Assay, Transfection, shRNA, Negative Control

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Impact of the C-X-C motif chemokine ligand 8/syndecan-1 (CXCL8/SDC1) axis on tumor initiation, growth, and metastasis of thyroid cancer (THCA) stem cells i n Vivo . (A) Diagram of the in vivo animal experiment protocol, with the green syringe representing anti-immunoglobulin G (anti-IgG) and anti-CXCL8 treatments. (B) Western blot analysis of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway-related proteins in subcutaneous tumor tissues of nude mice from each group. (C) Gross anatomy of subcutaneous transplant tumors in nude mice (left) and corresponding weight statistics (right). (D) Images of primary tumors in nude mice from each group (left) and diameter analysis (right). (E) Statistical analysis of axillary lymph node (LN) metastasis in subcutaneous transplant tumors of nude mice from each group. (F) Hematoxylin and eosin (H&E) staining to assess bone metastasis of primary tumors in nude mice from each group. (G) Images of lung metastasis in nude mice from each group (left) and diameter analysis (right). (H) H&E staining to evaluate lung metastasis in nude mice from each group. ∗ P < 0.05 compared between two groups, ∗∗ P < 0.01 with 6 nude mice in each group. p-JAK2: phosphorylated-JAK2; p-STAT3: phosphorylated STAT3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; S_sh-NC: FTC238-S cells transfected with short hairpin RNA-negative control (sh-NC); S_sh-SDC1: FTC238-S cells transfected with short hairpin RNA-SDC1 (sh-SDC1).

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: In Vivo, Western Blot, Staining, Transfection, shRNA, Negative Control

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Schematic diagram of the molecular mechanism by which monocytes promote self-renewal, migration, and invasion of thyroid cancer (THCA) stem cells through the C-X-C motif chemokine ligand 8/syndecan-1 (CXCL8/SDC1) axis. JAK2: Janus kinase 2; STAT3: signal transducer and activator of transcription 3.

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: Migration

Journal: iScience

Article Title: Single mutation tunes IRF4 function and mediates B cell character to ameliorate murine lupus

doi: 10.1016/j.isci.2025.113884

Figure Lengend Snippet: IRF4 K123R mutation moderated SLE depending on B7-H1 signaling (A–C) WT and IRF4 K123R B cells were stimulated with LPS for 24 h. (A) qPCR analysis of STAT3 and B7-H1. (B) Western blotting analysis of total/phosphorylated STAT3 and IRF4. (C) qPCR analysis of STAT3 and B7-H1 in B cells pretreated with or without STAT3 inhibitor. (D) B7-H1 expression in B cells pretreated with or without STAT3 inhibitor was analyzed by flow cytometry. (E–G) STAT3 inhibitor pretreated or untreated WT or IRF4 K123R B cells (4 × 10 7 ) and WT T cells (2 × 10 7 ) were intravenously injected into RAG1 −/− mice. ALD-DNA-activated BMDC was injected into the reconstituted mice through tail veil. (E). Serum anti-dsDNA IgG was measured by ELISA at week 20. (F). Proteinuria level was analyzed at week 20. (G). The deposition of IgG and C3 in renal sections was analyzed by immunofluorescence at week 20. (H) IRF4, STAT3, or phosphorylated STAT3 were pulled down from B cells by co-immunoprecipitation experiments, and western blotting was used to analyze indicated protein conjunctions. (I) Schematic of the working hypothesis. n = 5 in each group of reconstituted murine SLE model in (E–G). The results are presented as the mean ± SEM from three separate experiments. ns, no significance; ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001 using nonparametric Mann-Whitney tests. Scale bars, ×400: 50 μm.

Article Snippet: Blocking with 5% BSA in TBS, the membranes were incubated with antibodies against IRF4, STAT3, phosphorylated STAT3 and β-Actin (Cell Signaling Technology), followed by incubation with HRP conjugated secondary antibody (Cell Signaling Technology).

Techniques: Mutagenesis, Western Blot, Expressing, Flow Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Immunoprecipitation, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Effect of ruxolitinib on JAK/STAT signaling pathway-related genes and inhibition of interferon (IFN)-γ-Induced STAT1 activation by ruxolitinib. (A) Heatmap of transcription levels (fold-changes) of the JAK/STAT signaling-related genes in CCD841 and Jurkat cells after treatment with IFN-γ and ruxolitinib. (B) Nuclear extracts prepared from CCD841 cells with or without IFN-γ activation and analyzed for STAT1 activation using electrophoretic mobility shift assay (EMSA) with the STAT1-binding consensus sequence DNA probe. Ruxolitinib at 50, 250, and 500 nM was incubated with STAT1-DNA complexes. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Inhibition of STAT1 phosphorylation by ruxolitinib in immune cells and normal intestinal epithelial cells. (A) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in Jurkat, RAW and CCD841 cells treated with 50 IU/mL IFN-γ for the indicated times. (B–D) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (B) Jurkat, (C) RAW, and (D) CCD841 cells from (A) . (E) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in cells pretreated with IFN-γ (8 h) followed by Ruxolitinib (RUX, 24 h) at indicated concentrations (μM). (F–H) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (F) Jurkat, (G) RAW, and (H) CCD841 cells from (E) . # P < 0.05, ## P < 0.01, ### P < 0.001 vs. 0 μM RUX (IFN-γ-treated). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, * P < 0.001 vs. the group with IFN-γ concentration at 0; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the IFN-γ-treated group with RUX concentration at 0. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Concentration Assay