Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neutrophilic granule protein promotes lipopolysaccharide-induced iNOS/NO expression via JAK2/STAT1 signaling pathway and augments bacteria clearance of macrophages

doi: 10.1007/s00018-025-05865-9

Figure Lengend Snippet: NGP controls the expression of iNOS/NO by regulating the JAK2/STAT1 signaling pathway. ( A ) Ngp NC /RAW and Ngp HI /RAW cells were treated with LPS (10 μg/mL) for 6 h and lysed for RNA sequencing. The KEGG database was utilized to enrich signal pathways exhibiting significant differences. ( B ) Ngp NC /RAW and Ngp HI /RAW cells were stimulated with LPS for 6 h. The cells were harvested and subjected to CSP100 PLUS phosphorylation microarray analysis. ( C ) The Ngp NC /RAW and Ngp HI /RAW cells were treated without or with LPS for 6 and 12 h respectively. Western blotting analysis was performed to assess the phosphorylation levels of JAK2 and STAT1. ( D ) The Ngp NC /RAW and Ngp HI /RAW cells were treated with LPS for 6 h and subjected for IP-MS to screen for the binding protein of NGP and verify the interaction. ( E ) The Ngp WT /RAW cells were treated without or with LPS for 6 h, harvested for Co-IP, and STAT1 expression levels were quantified via grayscale intensity analysis. ( F – H ) The predicted structures of NGP and STAT1 were generated by Alphafold and subjected to GRAMM Web Server for molecular docking. ( F ) Molecular docking generic matching information. ( G ) Predicted binding sites between NGP and STAT1. ( H ) The protein–protein interaction cartoon model generated by PyMOL. (I-J) The phosphorylation levels in PBS- or LPS-treated Ngp NC /RAW and Ngp HI /RAW cells were examined by LSCM (Scale bar = 20 μm). (K-M) Ngp NC /RAW and Ngp HI /RAW cells were pre-treated without or with JAK2 inhibitor (10 μM), STAT1 inhibitor (50 μM), or control solution for 2 h and then stimulated without or with LPS for 12 h. The supernatants and cells were harvested for NO expression, Nos2 mRNA level and positive ratio of iNOS by aforementioned methods ( n = 3). (N–O) Ngp NC /RAW and Ngp. HI /RAW cells were transfected with scramble siRNA or STAT1 siRNA, respectively, followed by 12-h stimulation with PBS or LPS. The supernatants and cells were harvested for concurrent measurement of NO levels and Nos2 mRNA expression via Griess assay and qRT-PCR ( n = 3). The data represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one-way ANOVA to compare the results. * p < 0.05

Article Snippet: The JAK2 inhibitor (Ruxolitinib) and the STAT1 inhibitor (Fludarabine) were purchased from TargetMol (Boston, USA).

Techniques: Expressing, RNA Sequencing, Phospho-proteomics, Microarray, Western Blot, Protein-Protein interactions, Binding Assay, Co-Immunoprecipitation Assay, Generated, Control, Transfection, Griess Assay, Quantitative RT-PCR

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 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: 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: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A GPR4 regulates JAK2 and STAT3 phosphorylation via western blotting. n = 3. Scale bar: 100 µm. B Representative IF images of STAT3-Y705 phosphorylation regulated by GPR4. C Quantitative analysis of STAT3-Y705 phosphorylation in GPR4 - OE SW480 cell line and ( D ) GPR4 - KD HCT116 cell lline. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. E Static treatment reduces both pSTAT3-Y705 and the expression of LOXL2, COL1A1, and TGF-β in GPR4 - OE SW480 cells. F RT-PCR analysis for LOXL2 in SW480 cell lines after receiving Stattic treatment. Two-tailed t test, n = 3 biologically independent samples. G RT-PCR analysis for TGF-β in SW480 cell lines after receiving Stattic treatment. Data are presented as mean ± SD.Two-tailed t test, n = 3 biologically independent samples. H STAT3 transcription factor binding site. I In SW480 cells, STAT3 binds to the LOXL2 promoter region confirmed via ChIP assays. J STAT3 binds to the TGF-β promoter region confirmed via ChIP assays. K RT-PCR assesses the transcriptional activity of LOXL2 promoter regions and TGF-β promoter region L . Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: The LOXL2 inhibitor β-aminopropionitrile (BAPN), JAK2 inhibitor fedratinib (fedratinib hydrochloride hydrate), and STAT3 inhibitor Stattic were purchased from MedChemExpress (CAT: HY-Y1750, HY-10409A, and HY-13818).

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A Co-immunoprecipitation (Co-IP) experiments shows an endogenous interaction between GPR4 and JAK2 in SW480 cells and B HCT116 cells. n = 3. C , D Co-IP experiments shows an exogenous interaction between GPR4 and JAK2 in HEK293T cells. n = 3. E Representative IF images of colocalization between GPR4 and JAK2. n = 3. Scale bar: 19.4 µm. F Pearson’s correlation coefficients of colocalization in HCT116 cells (Pearson test) and G SW480 cells (Pearson test). H Full-length HA-JAK2, HA-tagged JAK2 FERM-SH2 domain (amino acids 1-510), HA-tagged JAK2 JH1-JH2 domain (amino acids 540-1124) and empty vector plasmid co-transfected with Full-length FLAG-tagged GPR4 plasmid in HEK293T cells, Co-IP assay was performed to verify their interaction. I Full-length Flag-GPR4 plasmid, the extracellular and 7-transmembrane domain (Flag-GPR4-1-290aa) plasmid, the intracellular C-terminal domain (Flag-GPR4-290-362aa) plasmid, or the extracellular, 7-transmembrane, partial intracellular hydrophobic segment (Flag-GPR4-1-330aa) plasmid, and empty vector plasmid co-transfected with Full-length HA-tagged JAK2 plasmid in HEK293T cells, Co-IP assay was performed to verify their interaction.

Article Snippet: The LOXL2 inhibitor β-aminopropionitrile (BAPN), JAK2 inhibitor fedratinib (fedratinib hydrochloride hydrate), and STAT3 inhibitor Stattic were purchased from MedChemExpress (CAT: HY-Y1750, HY-10409A, and HY-13818).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection

Journal: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A The dynamic changes with time for protein level of JAK2-Y1007/1008 and STAT3-Y705 stimulated by acidic environment in SW480 cells and B HCT116 cells. C The protein level of JAK2-Y1007/1008 and STAT3-Y705 in GPR4-KD HCT116 cells stimulated by acidic environment. D The protein level of JAK2-Y1007/1008 and STAT3-Y705 in GPR4-OE SW480 cells stimulated by acidic environment. E – G Representative IF images and quantitative analysis of TAT3-Y705 expression in HCT116 stimulated by acidic environment. Data are presented as mean ± SD. Scale bars, 100 µm. Two-tailed t test, n = 3 biologically independent samples. H – J Representative IF images and quantitative analysis of TAT3-Y705 expression in SW480 stimulated by acidic environment. Scale bars, 100 µm. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: The LOXL2 inhibitor β-aminopropionitrile (BAPN), JAK2 inhibitor fedratinib (fedratinib hydrochloride hydrate), and STAT3 inhibitor Stattic were purchased from MedChemExpress (CAT: HY-Y1750, HY-10409A, and HY-13818).

Techniques: Expressing, Two Tailed Test