Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 regulate casein synthesis in GMECs. (A, B) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, phosphorylated JAK2 and STAT5 after transfection with pcDNA3.1-ELF5 or pcDNA3.1-NC for 48 h. (C, D) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, p-JAK2, and p-STAT5 after transfection with siRNA-ELF5 or siRNA-NC (100 nM) for 48 h. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. * p<0.05, ** p<0.01. siRNA, small interfering ribonucleic acid; NC, negative control; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Expressing, Transfection, Quantitative Proteomics, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: The protein abundance of caseins after JAK2 and STAT5 inhibition in GMECs. (A) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, and phosphorylated JAK2 and STAT5 after JAK2 inhibitor Tyrphosting AG490 (30 μM) treatment for 48 h. (B) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, and p-STAT5 after STAT5 inhibitor STAT5-IN-1 (50 μM) treatment for 48 h. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. ** p<0.01. GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Quantitative Proteomics, Inhibition, Expressing

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 mediates casein synthesis by STAT5 activity in GMECs. (A) Cells were treated with STAT5-IN-1 (50 μM) or DMSO, followed by pcDNA3.1-ELF5 or pcDNA3.1-NC transfection for 48 h. (B) Cells were treated with STAT5-IN-1 (50 μM) or DMSO, followed by siRNA-ELF5 or siRNA-NC (100 nM) transfection for 48 h. The protein abundances of αS1-casein, αS2-casein, β-casein, κ-casein, and p-STAT5 were detected. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-STAT5 was normalized to total STAT5. Values are presented as mean±SEM. a–d Different lowercase letters represent significant differences (* p<0.05). siRNA, small interfering ribonucleic acid; NC, negative control; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Activity Assay, Transfection, Quantitative Proteomics, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 mediates casein synthesis by JAK2 and STAT5 activity in GMECs. (A) Cells were treated with Tyrphosting AG490 (30 μM) or DMSO, followed by pcDNA3.1-ELF5 or pcDNA3.1-NC transfection for 48 h. (B) Cells were treated with Tyrphosting AG490 (30 μM) or DMSO, followed by siRNA-ELF5 or siRNA-NC (100 nM) transfection for 48 h. The protein abundances of αS1-casein, αS2-casein, β-casein, κ-casein, p-JAK2, and p-STAT5 were detected. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. a–d Different lowercase letters represent significant differences (* p<0.05). NC, negative control; siRNA, small interfering ribonucleic acid; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Activity Assay, Transfection, Quantitative Proteomics, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: The interaction between ELF5 and STAT5 in GMECs. (A) Cells were transfected with ELF5-MYC and STAT5-FLAG for 48 h. Then, total protein of cells was extracted and incubated with MYC (or FLAG) tag antibody and protein G magnetic beads at 4°C overnight. The protein abundance of ELF5 and STAT5 was detected. (B) The mRNA expression of STAT5a and ELF5 gene was measured after pCMV-STAT5a and pCMV-NC transfection for 48 h. Values are presented as mean±SEM. ** p<0.01. GMECs, goat mammary epithelial cells; NC, negative control; SEM, standard error of the mean.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Transfection, Incubation, FLAG-tag, Magnetic Beads, Quantitative Proteomics, Expressing, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: Molecular mechanism of ELF5 promotes casein synthesis by enhancing the activity of JAK2/STAT5 signaling pathway in goat mammary epithelial cells. PRLR stands for prolactin receptor.

Article Snippet: For the inhibition of JAK2/STAT5 pathway, JAK2 inhibitor AG490 (30 μM, MedChemExpress) or STAT5 inhibitor STAT5-IN-1 (50 μM, MedChemExpress) were treated with cells.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: IRG1/itaconate/NRF2/GSH axis in tumor-associated macrophages drives therapy resistance and immune evasion in BRCA1-deficient breast cancer

doi: 10.1101/2025.10.14.682312

Figure Lengend Snippet: a , Schematic representation of the metabolic pathway of IRG1-related metabolites. b, Heatmap indicates the relative levels of itaconate and its related metabolites in CM derived from BP-TAM and control macrophages in the untargeted metabolomic analysis. c, RT-qPCR analysis of IRG1 expression in THP-1-Mφ and 436-TAMs. Data are presented as mean ± s.d.; unpaired Student’s t-test; n = 3. d, Western blot analysis of IRG1 protein expression in THP-1-Mφ and 436-TAMs. e, RT-qPCR analysis of IRG1 expression in BMDMs and BP-TAMs. Data are presented as mean ± s.d.; unpaired Student’s t-test; n = 3. f, Western blot analysis of IRG1 protein expression in BMDMs and BP-TAMs. g, GSEA of RNA-seq data from BMDMs and BP-TAMs. h,i, Western blot analysis of STAT5 and phosphorylated STAT5 (p-STAT5) expression in THP-1-Mφ, 436-TAMs, BMDMs, and BP-TAMs. j, Western blot analysis of IRG1 protein expression in THP-1-Mφ and 436-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. The inhibitors were used at the following concentrations: STAT3-IN-1 (10 μM), STAT5-IN-1 (100 μM), and pyrrolidinedithiocarbamate ammonium (PDTC, 10 μM). k, RT-qPCR analysis of IRG1 mRNA expression in THP-1-Mφ and 436-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. Fold changes and P values in IRG1 mRNA levels in TAMs relative to THP-1-Mφ are shown. The inhibitors were used at the same concentrations as in ( j ). l, Western blot analysis of IRG1 protein expression in BMDMs and BP-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. The inhibitors were used at the same concentrations as in ( j ). m, RT-qPCR analysis of IRG1 mRNA expression in BMDMs and BP-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. Fold changes and P values in IRG1 mRNA levels in TAMs relative to BMDMs are shown. The inhibitors were used at the same concentrations as in ( j ).

Article Snippet: The following small-molecule compounds, metabolites, inhibitors, and neutralizing antibodies were used in this study: olaparib (HY-10162, MedChemExpress), L-glutathione reduced (GSH; G4251, Sigma-Aldrich, St. Louis, MO, USA), N-acetyl-L-cysteine (NAC; A7250, Sigma-Aldrich), L-(+)-lactic acid (L1750, Sigma-Aldrich), sodium L-lactate (71718, Sigma-Aldrich), itaconic acid (T4837, TargetMol, Wellesley Hills, MA, USA), citraconic acid (C82604, Sigma-Aldrich), 4-octyl itaconate (4-OI; SML2338, Sigma-Aldrich), dimethyl itaconate (DMI; T5377, TargetMol), dimethyl citraconate (DMC; C0346, TCI America, Portland, OR, USA), DL-buthionine-sulfoximine (BSO; 19176, Sigma-Aldrich), sodium oxamate (LDHi; O2751, Sigma-Aldrich), STAT5-IN-1 (S6784, Selleck Chemicals, Houston, TX, USA), STAT3-IN-1 (S0818, Selleck Chemicals), pyrrolidinedithiocarbamate ammonium (PDTC; S3633, Selleck Chemicals), RSL3 (S8155, Selleck Chemicals), Ferrostatin-1 (Fer-1; S7243, Selleck Chemicals), ML385 (T4360, TargetMol), and IRG1-IN-1 (T78525, TargetMol).

Techniques: Derivative Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, RNA Sequencing

Journal: bioRxiv

Article Title: IRG1/itaconate/NRF2/GSH axis in tumor-associated macrophages drives therapy resistance and immune evasion in BRCA1-deficient breast cancer

doi: 10.1101/2025.10.14.682312

Figure Lengend Snippet: STAT5-dependent activation of the IRG1/itaconate pathway in TAMs reprograms mitochondrial metabolism and activates NRF2-driven GSH biosynthesis. The TAM-derived GSH protects tumor cells from ROS-induced DNA damage and ferroptosis, while concurrently suppressing STING-mediated immune activation in both tumor and dendritic cells. Pharmacological inhibition of IRG1 restores tumor sensitivity to PARPi, reactivates anti-tumor immunity, and significantly suppresses tumor growth in BRCA1-deficient preclinical models.

Article Snippet: The following small-molecule compounds, metabolites, inhibitors, and neutralizing antibodies were used in this study: olaparib (HY-10162, MedChemExpress), L-glutathione reduced (GSH; G4251, Sigma-Aldrich, St. Louis, MO, USA), N-acetyl-L-cysteine (NAC; A7250, Sigma-Aldrich), L-(+)-lactic acid (L1750, Sigma-Aldrich), sodium L-lactate (71718, Sigma-Aldrich), itaconic acid (T4837, TargetMol, Wellesley Hills, MA, USA), citraconic acid (C82604, Sigma-Aldrich), 4-octyl itaconate (4-OI; SML2338, Sigma-Aldrich), dimethyl itaconate (DMI; T5377, TargetMol), dimethyl citraconate (DMC; C0346, TCI America, Portland, OR, USA), DL-buthionine-sulfoximine (BSO; 19176, Sigma-Aldrich), sodium oxamate (LDHi; O2751, Sigma-Aldrich), STAT5-IN-1 (S6784, Selleck Chemicals, Houston, TX, USA), STAT3-IN-1 (S0818, Selleck Chemicals), pyrrolidinedithiocarbamate ammonium (PDTC; S3633, Selleck Chemicals), RSL3 (S8155, Selleck Chemicals), Ferrostatin-1 (Fer-1; S7243, Selleck Chemicals), ML385 (T4360, TargetMol), and IRG1-IN-1 (T78525, TargetMol).

Techniques: Activation Assay, Derivative Assay, Inhibition

Journal: Frontiers in Medicine

Article Title: The research on cycloastragenol in the treatment of brain metastases from lung cancer: mechanistic exploration of radiotherapy sensitization and amelioration of brain injury

doi: 10.3389/fmed.2025.1616894

Figure Lengend Snippet: CAG inhibits neutrophil chemoattraction-related NF-κB and STAT signaling pathway activity. (A,B) Molecular simulation diagrams and interaction tables between CAG and the STAT5B receptor; (C–E) ELISA was used to analyze the effects of CAG and STAT5 inhibitor on the expression of TGF-β1, CXCL3, and CCL5 in LLC cells (N = 3). (F–H) qPCR analysis of the effects of CAG and STAT5 inhibitor on the expression of TGF-β1, CXCL3, and CCL5 in LLC cells ( N = 3). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: (1) Cycloastragenol (source leaf), physiological sodium chloride solution (Fengyuan), T-25, T-75 sterile culture bottle, Trypsin (Biosharp), 1% streptomycin (Biosharp), 4% paraformaldehyde (Biosharp), 1 × PBS (Biosharp), DMEM medium (Gibco), 1,640 medium (Gibco), Fetal bovine serum (ExCell), Puromycin (Biosharp) pasteurized straw, 1 mL disposable sterile syringe, 10 mL round-bottomed capped centrifuge tube (Biosharp), 1.5 mL sharp-bottomed centrifuge tube (Biosharp), STAT5 inhibitor (STAT5-IN-1, HY-101853, MCE). (2) Ki67 (Servicebio, GB111141 ), CD31 (Servicebio, GB11063-1), Ly6G (Servicebio, GB11229), Iba-1 (PTG, 26177-1-ap), CD16 (ABCam, ab183685), CD206 (CD206, ab182422) was used for IF. (3) Mouse kits for ELISA: TGF-β1 (ruixinbio RX104768H), CXCL3 (ruixinbio RX101460H), CCL5 (ruixinbio RX105344H). (4) PCR reagent: RNA extract (Xavier, G3013), Chloroform substitute (Xavier, G3014), Isopropanol (Xavier, 80109218), Reverse Transcription Kit (Xavier, G3337). (5) Antibodies required for brain transplantation tumor experiment: InVivoMab anti-mouse Ly6G (BE0075-1.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Scientific Reports

Article Title: IL-15-Activated CD38 + HLA-DR + CD8 + T cells induce liver injury in cirrhosis via JAK/STAT5 and PI3K/mTOR pathways

doi: 10.1038/s41598-025-02693-6

Figure Lengend Snippet: The signal pathways involved in IL-15-induced innate cytotoxicity of CD38 + HLA-DR + CD8 + T cells. (A) 1 × 10 6 CD8 + T cells from healthy donors were stimulated with IL-15 (20 ng/mL) for 48 h and 72 h, after which phosphorylation of signaling proteins was assessed by flow cytometry for STAT5 ( n = 10), ERK1/2 ( n = 7) and mTOR ( n = 8). Representative dot plots and the summary data show the expression of signaling proteins in CD38 + HLA-DR + CD8 + T cells. (B) The percentage of CD38 + HLA-DR + CD8 + T cells was analyzed after inhibitors treatment ( n = 4). Representative dot plots from a single donor (left) and summary data (right) are presented. (C-G) The percentage NKG2D, FasL, perforin, and Granzyme B in CD38 + HLA-DR + CD8 + T cells were analyzed after inhibitors treatment ( n = 4). (H) CD8 + T cells from healthy donors were pre-treated with STAT5 inhibitor pimozide, MEK inhibitor PD98059, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) stimulation for the next 72 h. Then, CD8 + T cells were co-cultured with CFSE-labeled K562 cells at a 10:1 E: T ratio and cytotoxicity against K562 cells evaluated ( n = 4). Representative dot plots and the summary data show expression of PI in the gate of CFSE + . (I) Schematic representation of cytokine-mediated crosstalk between T cells and liver cells in liver cirrhosis. The Wilcoxon matched-pairs signed rank test (A) was used for comparisons among groups. The one-way ANOVA (B , C , D-H) was used for comparisons between groups. Control group was indicated treatment only IL-15. ns, not significant, * p < 0.05, ** p < 0.01.

Article Snippet: CD8 + T cells (1 × 10 6 ) from healthy donors were pre-treated with STAT5 inhibitor pimozide, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) (Thermo, MA, USA) stimulation for the next 72 h. All the inhibitors were obtained from MedChemExpress (NJ, USA).

Techniques: Phospho-proteomics, Flow Cytometry, Expressing, Cell Culture, Labeling, Control