Journal: The Journal of Endocrinology

Article Title: Estrogen accelerates heart regeneration by promoting the inflammatory response in zebrafish

doi: 10.1530/JOE-19-0413

Figure Lengend Snippet: Estrogen induces inflammation in the injured zebrafish heart. A. Gene Ontogeny (GO) terms significantly enriched in genes differentially expressed in female vs male hearts at 7 dpc, n = 3. (B) Comparison of gene expression patterns at 7 dpc in the hearts of female vs male, E2 (1 nM)-treated male vs untreated male, and tamoxifen (1 μM)-treated female vs untreated female, n = 3. (C) Expression of immune and inflammation-related genes in the datasets, n = 3. (D, E and F) Expression of ifn-γ at 7dpc in female and male zebrafish heart and uninjured fish (D). Tamoxifen decreased (E) and E2 increased (F) the expression of ifn-γ in females and males at 7 dpc. n = 4, two-tail t test , * P < 0.05, ** P < 0.01, *** P < 0.001. (G and H) Western blot of STAT1 and STAT3 in female and male heart at 7 dpc using different treatments. H, bar chart showing the quantification of STAT1 expression in male fish in panel G, n = 3, two-tail t test , * P < 0.05.

Article Snippet: The following primary antibodies were used: mouse anti-GAPDH (60004-1; Proteintech, Rosemont, IL, USA) at 1:10000, mouse anti-zebrafish vitellogenin, JE-2A6 (V01408102; Biosense, Bergen, Norway) at 1:2000, mouse anti-embCMHC (N261.1, DSHB, Iowa City, IA, USA) at 1:500, rabbit anti-STAT1 and rabbit anti-STAT3 (R1408-2 and ET1607-38, both from Huabio, Hangzhou, China) at 1:2000.

Techniques: Expressing, Western Blot

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Overview of all differentially expressed genes categorized according to their activity in cancer biology.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Validation of differential gene expression in MCC cell lines on protein level and mRNA level. (A) TPM values were acquired from our sequencing output. The data represent mean values ± SEM from three independent experiments (two for MKL-1 control). (B) After 72 h of treatment with or without IFNγ (3,000 U/ml), the Merkel cell carcinoma cell lines MKL-1, MKL-2, and WaGa were stained for signal transducer and activator of transcription 1-alpha/beta (STAT1), bone marrow stromal antigen 2 (BST2), C-X-C motif chemokine 10 (CXCL10), and HLA class II histocompatibility antigen gamma chain (CD74) and analyzed by flow cytometry. The average MFI of three independent experiments ± standard error is indicated. p -values were determined using the paired student’s t -test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Biomarker Discovery, Gene Expression, Sequencing, Control, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Pro- and anti-inflammatory macrophages adjust UCP2 protein levels based on their intrinsic metabolism and available metabolites

doi: 10.1101/2025.09.08.674165

Figure Lengend Snippet: (A) Scheme of differentiation and polarization of bone marrow-derived macrophages into pro- and anti-inflammatory phenotypes. (B) Representative WB showing STAT1 and STAT6 as well as their phosphorylated forms (pSTAT1 and pSTAT6) in LPS-MΦ and IL4-MΦ, respectively. (C) Representative WB confirming the expression of UCP2 in pro-ad anti-inflammatory macrophages. Recombinant mouse UCP2 (1 ng) and spleen, and skeletal muscle (SkM) were used as positive and negative controls for UCP2 expression, respectively. 20 µg total cell or tissue protein was loaded per lane.

Article Snippet: Antibodies against STAT1 (9172, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT1 (9167, Cell Signaling Technology, Inc., Massachusetts, USA), STAT6 (9162, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT6 (9361, Cell Signaling Technology, Inc., Massachusetts, USA), bcl2 (3498, Cell Signaling Technology, Inc., Massachusetts, USA), BAX (2772, Cell Signaling Technology, Inc., Massachusetts, USA), NFKBIA/IkB alpha (H-4) (sc-1643, Santa Cruz Biotechnology Inc., USA), and α-tubulin (801202, Bio Legend Inc., San Diego, USA) were used at dilutions 1:1000, and 1:10000, respectively.

Techniques: Derivative Assay, Expressing, Recombinant

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Molecular docking and dynamic simulations of ROF and JAKs/STATs signaling molecules (A) The binding modes of ROF toward JAK2, JAK3, STAT1, and STAT3. (B–F) The dynamic simulations of JAK2-ROF and JAK3-ROF complexes. The HBond (B), RMSD curves (C), RMSF values (D), Rg values (E), and SASA (F) were analyzed by GROMACS.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Binding Assay

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on the JAK2/3-STAT1/3 signaling pathways in Con A-induced hepatitis (A) The phosphorylated levels of JAK2, STAT1, and STAT3 were measured in hepatic tissues using IHC staining ( n = 3; scale bar = 40 μm). (B) The IODs of each protein were detected by using ImageJ. (C) The JAK2/3-STAT1/3 signaling molecules were measured in hepatic tissues with Western blot analysis ( n = 3). The gray value analysis was standardized using the intensity of β-actin and is depicted in bar graphs. The results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs the Con A group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Protein-Protein interactions, Immunohistochemistry, Western Blot

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on Th1/Th17 cells differentiation in vitro (A) The purity of isolated CD4 + T cells was measured by flow cytometry. (B) The viability of CD4 + T cells following 5–20 μM ROF treatments for 24 h was assessed utilizing the CCK-8 assay. (C, D) CD4 + T cells were pre-exposed for 1 h to ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM), followed by stimulation with Con A for 24 h. The levels of p-JAK2, p-JAK3, p-STAT1, and p-STAT3 were monitored by Western blotting and normalized to each protein’s total concentration in the graphs. (E) The generation of IFN-γ or IL-17 in the cellular supernatants of Th1 or Th17 subtypes were measured using ELISA. (F) The gene expression levels of T-bet or RORγt in differentiated T cells were evaluated by PCR. The values are presented as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, Isolation, Flow Cytometry, CCK-8 Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on primary hepatocytes apoptosis in vitro (A) Primary hepatocytes were pretreated with ROF (5, 10, 20 μM) for 6 h and treated with or without Con A (10 μg/mL) for 24 h, the cell viability of each group was analyzed by CCK-8 assay. (B) Flow cytometry analysis of apoptosis in primary hepatocytes. (C) Primary hepatocytes were pretreated with ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM) for 6 h, and subsequently triggered with Con A (10 μg/mL) for 24 h. The IL-6, p-JAK2, p-STAT1, p-STAT3, BNIP3, and Bax expression levels were evaluated by using Western blotting. (D) The quantification and standardization of the protein expression were performed using ImageJ. The results are presented as mean ± SEM of three independent experiments. ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: Clinical Oral Investigations

Article Title: Leptin reduces in vitro cementoblast mineralization and survival as well as induces PGE2 release by ERK1/2 commitment

doi: 10.1007/s00784-020-03501-3

Figure Lengend Snippet: Kinetic analysis performed on OCCM-30 cells after stimulation with leptin. The expression of SHP2; COX2; p-STAT1; STAT1; p-STAT3; STAT3; p-ERK1/2; ERK1/2; p-P38; P38; p-JNK; and JNK were visualized by western blot: Leptin upregulates p-STAT1; p-STAT3; and p-ERK1/2 as well as COX2 on OCCM cells whereas no activation of P38 or JNK was observed. Each experiment was repeated three times

Article Snippet: Membranes were blocked with 5% non-fat milk (T145.1, ROTH) and incubated for 1 h at room temperature employing the following antibodies: leptin receptors (ObR) (ab5593, Abcam); ERK1/2 (MBS8241746, BIOZOL), dilution 1:1000; phospho-ERK1/2 (44-680G, Thermo-Fisher) dilution 1:1000; JNK (MBS840351, BIOZOL) dilution 1:500; phospho-JNK (07-175, Thermo-Fisher) dilution 1:500; P38 MAPK (9212, Cell Signaling Technology) dilution 1:1000; phospho-P38 MAPK alpha (MA5-15182, Thermo-Fisher) dilution 1:500; STAT1 (AHP2527, Bio-Rad) dilution 1:1000; phospho-STAT1 Tyr701 (05-1064, Thermo-Fisher) dilution 1:1000; phospho-STAT1 S727 (ab109461, Abcam) dilution 1:1000; STAT3 (PA1-86605, Thermo-Fisher) dilution 1:1000; phospho-STAT3 Ser727 (OPA1-03007, Thermo-Fisher) dilution 1:500; Cytochrome C (ab65311, Abcam) dilution 1:1000; cPLA2α (orb100010, BIOZOL) dilution 1:1000; cPLA2β (ab198898, Abcam) dilution 1:1000; SHP2 (PA5-27312, Thermo Fisher) dilution 1:1000; COX 2 (ab62331, Thermo-Fisher) dilution 1:1000.

Techniques: Expressing, Western Blot, Activation Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Overexpression of NLRP12 enhances macrophage immune response and alleviates herpes simplex keratitis

doi: 10.3389/fcimb.2024.1416105

Figure Lengend Snippet: NLRP12 enhances antiviral functions through IL-18 downstream JAK-STAT signaling. (A) Protein levels of STAT1, p-STAT1, STAT4, pSTAT4 were assessed at 8h, 12h and 24h post-infection in both groups. Western blot bands were normalized to β-actin and compared against controls (n=3 per group). (B) Immunofluorescence images demonstrating MHC II + cell staining. All of the data are representative of at least three independent experiments. Data are presented as the mean ± SEM. Statistical differences were determined using two-way ANOVA (A) . ns, not significnat, *P <0.05, ***P <0.001, ****P <0.0001.

Article Snippet: The membranes were then blocked with Y-Tec 5min Rapid Blocking Buffer (YWB0501, Yoche, China) and incubated overnight with primary antibodies against NLRP12 (1:1000, YT7568, Immunoway, USA), HSV-gB (1:500, sc-56987, Santa Cruz, USA), HSV-gD (1:500, sc-21719, Santa Cruz, USA), CASP1 (1:2000, 81482–1-RR, Proteintech, China), N-GSDMD (1:2000, ab215203, Abcam, UK), clv-CASP1 (1:500, AY0406, Abways, China), IL-18 (1:500, A1115, ABclonal, China), IL-1β (1:1000, ab254360, Abcam, UK), STAT1 (1:1000, CY5227, Abways, China), STAT1 (Phospho-S727) (1:1000, BM4541, Boster, USA), STAT4 (1:1000, BA0622–2, Boster, USA), Phospho-STAT4 (Tyr693) (1:1000, CY6503, Abways, China), and β-actin (1:20000, 81115–1-RR, Proteintech, China).

Techniques: Infection, Western Blot, Immunofluorescence, Staining