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: 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: 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

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: List of primer sequences used for RT-PCR analysis.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques:

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: Increased activity of STAT3, which is required for T-cell suppression. (a 1 ) Neutrophils before and after induction were tested for STAT1, STAT3, STAT5, p-STAT1, p-STAT3, and p-STAT5 expression by WB. (a 2 ) Grayscale analysis of WB results by image J software; the result was expressed as the gray value of the target band/the gray value of the reference protein. Increased expression levels of STAT3 and p-STAT3 proteins were found in neutrophils AI compared with those before induction. (b) Changes of mRNA expression of STAT1, STAT3, and STAT5 in neutrophils before and after induction. The mRNA level of STAT3 in neutrophils AI was significantly higher than that in neutrophils before induction. (c 1 and c 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h; T-cell proliferation was evaluated by CFSE labeling, and unstimulated T cells were used as a negative control. The samples were analyzed by flow cytometry. Ruxolitinib addition almost completely abolished the ability of CD10 − neutrophils to suppress T-cell proliferation. (c 3 ) Representative flow cytometry data of T-cell proliferation from one individual in coculture and indirect coculture systems. The red area represented the proliferating T-cell fraction. (d 1 and d 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h, and apoptotic T cells were assessed by flow cytometry. CD3 + T cells cultured alone were used as a negative control. Ruxolitinib addition almost completely prevented T-cell apoptosis. (d 3 ) Representative flow cytometry data of CD3 + T-cell apoptosis experiment from one individual in the coculture or indirect coculture system. CD3 + T cells without PI or annexin V were used as a blank control. Apoptotic T cells were marked as CD3 + /annexin + /PI + . (e 1 ) WB analysis of the changes in STAT3 and p-STAT3 expression before and after ruxolitinib addition in the induced neutrophil population and (e 2 ) grayscale analysis of WB results by imageJ software. Ruxolitinib inhibited the expression of STAT3 and p-STAT3 proteins. (f) Extracellular production of Arg-1 protein was evaluated by ELISA at 48 h before and after the addition of ruxolitinib in neutrophils AI . Significantly decreased expression of extracellular Arg-1 in neutrophils AI treated with ruxolitinib. (g) Changes in intracellular Arg-1 fluorescence intensity were detected by flow cytometry at 48 h before and after ruxolitinib addition in neutrophils AI . Significantly decreased expression of intracellular Arg-1 in neutrophils AI treated with ruxolitinib, mainly in the CD10 − neutrophil subset. (h) Arg-1 activity was determined at 48 h before and after ruxolitinib addition in neutrophils AI . A significant decrease in Arg-1 activity was observed in the cells treated with ruxolitinib. (i) mRNA expression of Arg-1 was detected by RT-PCR in neutrophils AI at 48 h before and after ruxolitinib addition. A significant decrease was observed in the mRNA expression of Arg-1 in the cells treated with ruxolitinib. (j) mRNA expression of STAT3 was detected by RT-PCR in neutrophils at 48 h before and after ruxolitinib addition. The expression of STAT3 gene in CD10 − neutrophil subpopulation significantly decreased. Each experiment was repeated 3 times. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ✶ ✶ ✶ ✶ P < 0.0001, ns: P > 0.05. Ctrl: Neutrophils before induction, neutrophils AI : Neutrophils after induction, WB: Western blot, CFSE: Carboxyfluorescein succinimidyl ester, CD10 − neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 − cells, CD10 + neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 + cells.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques: Activity Assay, Expressing, Software, Labeling, Negative Control, Flow Cytometry, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot