Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. CD163 surface expression in PBS or Kp52145-infected pBMDMs by flow cytometry. Values are shown as standard error of mean of two independent experiments in duplicate. **, p<0.01 determined by unpaired Student’s -test. B. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. cd163 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. D. Arginase-1 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. E. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 and the isogenic cps mutant, strain 52145-Δ wca K2 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. F. cd163 levels in pBMDMs non-infected (ni) or infected with the cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. G. arginase-1 levels in pBMDMs non-infected (ni) or infected with cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. In panels C, and D, ***p<0.001, **p<0.01, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Expressing, Infection, Flow Cytometry, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM/ 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. B. Immunoblotting analysis of phosphorylation of STAT3 (PSTAT3) and STAT3 in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. Immunoblotting analysis of phosphorylations of ERK (pERK), p38 (Pp38), and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. D. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with p38 inhibitor (SB203580, Tocris, 10 μg/mL, 2 h prior to infection), ERK inhibitor (U0126, LC laboratories, 20 μg/mL, 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. In panels A and D, ****p < 0.0001, ***p < 0.001, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Infection, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Opposing control of rhabdomyosarcoma growth and differentiation by myogenin and interleukin 4

doi: 10.1158/1535-7163.mct-08-0678

Figure Lengend Snippet: Figure 4. A, forced expression of myogenin inhibited RD/12 cell migra- tion and blocked directed migration toward IL-4. Columns, mean number of cells migrated in the lower compartment containing medium with or without IL-4, four independent replicates; bars, SE. Migration of RD/ 12-Myog cells was significantly lower than parental RD/12 cells (P < 0.001, Student's t test); IL-4 significantly (stars, P < 0.05, Student's t test) increased migration only of RD/12 and RD/12-Neo cells, but not of RD/12-Myog and RD/18 cells expressing high myogenin levels. IL-4 re- ceptor level (B) and signaling (C) in rhabdomyosarcoma cells with forced myogenin expression. Total proteins (100 μg) were loaded for the evalua- tion of total-STAT6, phospho-STAT6 (STAT6 P), IL-4 Rα, and IL-13 Rα ex- pression. Actin (15 μg of total proteins) was used as a housekeeping protein for sample normalization. M, marker of molecular weight.

Article Snippet: Mouse antihuman IL-13 Rα1 monoclonal antibody (clone 419718, 2 μg/mL), goat anti-human IL-4 Rα antibody (0.2 μg/mL), rabbit anti–phospho-STAT6 (Y641) antibody (0.5 μg/mL; R&D Systems), rabbit anti-total STAT6 (1.5 μg/mL, M-20; Santa Cruz Biotechnology), and rabbit anti-actin (Sigma) were used as primary antibodies.

Techniques: Expressing, Migration, Marker, Molecular Weight

Journal: Respiratory Research

Article Title: Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis

doi: 10.1186/s12931-024-02815-0

Figure Lengend Snippet: The effects of melatonin on abnormal polarization of pulmonary macrophages in AECOPD. a , b Mouse lung tissue sections from each group were probed with the specific antibody against pulmonary macrophage marker F4/80 (red) and co-probed with antibodies against M1 marker (iNOS) or M2 marker (CD206), and, representative lung immunofluorescence staining of lung tissue sections were shown (original magnification × 100 and × 500). c , d Western blot analysis of the expression of total-STAT1, phospho-STAT1, total-STAT6, phospho-STAT6, iNOS and Arg1 to β-tublin in lung tissue homogenates from air group, COPD (CS exposure) group, AECOPD (smoke + H3N2) group, AECOPD + melatonin (Mel, 30 mg/kg) group. Data expressed as mean ± SD ( n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were incubated with primary antibodies: total-STAT1 (1:1000, PTM-5754, PTM Bio, Hangzhou, China), Phospho-STAT1 (1:1000, 340797, ZenBio, Chengdu, China), total-STAT6 (1:1000, 380957, ZenBio), Phospho-STAT6 (1:500, sc-136019, Santa Cruz), iNOS (1:1000, ab178945, Abcam), Arg1 (1:1000, #93668S, Cell signaling technology), Caspase1 (1:500, sc-392736, Santa Cruz), MT1/2 (1:500, sc-398788, Santa Cruz), β-Tublin (1:5000, M20005, Abmart, Shanghai, China) and GAPDH (1:5000, ab181602, Abcam) overnight at 4℃.

Techniques: Marker, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Respiratory Research

Article Title: Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis

doi: 10.1186/s12931-024-02815-0

Figure Lengend Snippet: The effects of melatonin abnormal polarization of IAV-infected CSE-stimulated macrophages. a - c Western blot analysis of the expression of total-STAT1, Phospho-STAT1, total-STAT6, Phospho-STAT6 and IAV-NP to GAPDH as well as iNOS, Arg1 and MT1/2 to GAPDH in CSE-stimulated Raw264.7 cells infected by IAV/H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (10 μM, 100 μM and 200 μM, 3 h before H3N2 infection). d Representative Immunofluorescence images of iNOS (green) and Arg1 (red) expression in Raw264.7 cells infected by H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (200 μM, 3 h before H3N2 infection) (bar = 10 μm, original magnification × 630). e Quantitative RT-PCR measurements of the relative mRNA levels of MCP1, TNF-α, Arg1 and Fizz1 in CSE-stimulated Raw264.7 cells. Data expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001

Article Snippet: The membranes were incubated with primary antibodies: total-STAT1 (1:1000, PTM-5754, PTM Bio, Hangzhou, China), Phospho-STAT1 (1:1000, 340797, ZenBio, Chengdu, China), total-STAT6 (1:1000, 380957, ZenBio), Phospho-STAT6 (1:500, sc-136019, Santa Cruz), iNOS (1:1000, ab178945, Abcam), Arg1 (1:1000, #93668S, Cell signaling technology), Caspase1 (1:500, sc-392736, Santa Cruz), MT1/2 (1:500, sc-398788, Santa Cruz), β-Tublin (1:5000, M20005, Abmart, Shanghai, China) and GAPDH (1:5000, ab181602, Abcam) overnight at 4℃.

Techniques: Infection, Western Blot, Expressing, Immunofluorescence, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Phenotypic polarization of activated astrocytes: the critical role of lipocalin-2 in the classical inflammatory activation of astrocytes.

doi: 10.4049/jimmunol.1301637

Figure Lengend Snippet: FIGURE 7. LCN2 inhibition of IL- 4–induced STAT6 phosphorylation and its downstream gene expressions in astrocytes. Primary astrocyte cultures were treated with IL-4 (10 ng/ml), recombinant LCN2 protein (10 mg/ml), or leflunomide (10 mM) (30 min for Western blot analysis; 8 h for real-time RT-PCR; and 24 h for flow cytometric analysis). (A) Levels of phosphorylated STAT6 (pSTAT6 at Tyr641) and total STAT6 protein were evaluated by Western blotting. (B) The mRNA lev- els of alternative activation-related genes were quantified by real-time RT-PCR. Gapdh was used as an internal control. (C) ARG1 and MRC1 protein expres- sion was evaluated by flow cytometry. Mean fluorescence intensity (MFI) val- ues are also shown. Negative control is the measurement without specific Ab or with a control Ab. Results are mean 6 SD (n = 3). *p , 0.05, versus untreated controls or between the treatments in- dicated, #p , 0.05, versus IL-4 alone.

Article Snippet: Membranes were blocked with 5% skim milk, sequentially incubated with primary Abs (rabbit polyclonal anti-GFAP Ab, mouse monoclonal anti–a-tubulin [Sigma-Aldrich], rabbit polyclonal anti–phospho-STAT6 at Tyr641 Ab [Cell Signaling Technology, Beverly, MA], rabbit polyclonal anti-total STAT6 Ab [Abcam, Cambridge, MA]) and HRP-conjugated secondary Abs (anti-rabbit and anti-mouse IgG Ab; Amersham Biosciences), and proteins were detected by ECL (Amersham Biosciences).

Techniques: Inhibition, Phospho-proteomics, Recombinant, Western Blot, Quantitative RT-PCR, Activation Assay, Control, Cytometry, Negative Control

Journal: Cell reports

Article Title: Metallothionein 3 Controls the Phenotype and Metabolic Programming of Alternatively Activated Macrophages

doi: 10.1016/j.celrep.2019.05.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For total STAT6 and pSTAT6 (BD Biosciences) analysis, WT and Mt3 −/− Mϕ were stimulated with IL-4 for 15 min; for total STAT1 and pSTAT1 (Abcam) analysis, Mϕ were stimulated with 10 ng/ml IL-4 for 24h, followed by change of media and 5 ng/ml IFNγ for 15 min. To determine the translocation of HIF, total cytosolic and nuclear proteins were isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents according to manufacturer’s instructions (Thermo Scientific).

Techniques: Cell Culture, XF Assay, Plasmid Preparation, Transgenic Assay, Filtration, Cell Based Assay, Cytotoxicity Assay, Cell Viability Assay, Isolation, Software

Journal: BMC Complementary Medicine and Therapies

Article Title: Anti-cancer effects of genistein supplementation and moderate-intensity exercise in high-fat diet-induced breast cancer via regulation of inflammation and adipose tissue metabolism in vivo and in vitro

doi: 10.1186/s12906-025-04968-x

Figure Lengend Snippet: Effects of GEN, myokines, or their combination on the JAK1/STAT6 pathway in U937 cells. ( A ) U937 cells were treated with GEN, Iri (20 nM), or their combination after PMA and IL-4 treatment. ( B ) U937 cells were treated with GEN, OSM (20 ng/mL), or their combination after PMA and IL-4 treatment. ( a ) Representative pictures were shown. Protein expressions of ( b ) p-JAK1/JAK1 and ( c ) p-STAT6/STAT6 were assessed by Western blot assay. All data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the Newman-Keuls post hoc test. Statistical significance among groups was denoted by different alphabetical letters ( P < 0.05). GEN, genistein; H, breast cancer-induced mice fed a high-fat diet; HG, breast cancer-induced mice fed a high-fat diet with GEN; HE, breast cancer-induced mice fed a high-fat diet with moderate-intensity exercise; HGE, breast cancer-induced mice fed a high-fat diet with GEN and moderate-intensity exercise

Article Snippet: The primary antibodies were used in this study as follows: Bcl-2, Bax, Proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3, total-JAK1, phospho-JAK1, total-STAT6, phospho-STAT6 (Cell Signaling Technology, Danvers, MA, USA), Arg1 (Proteintech, Manchester, UK), CD163, CD68, and β-actin (Abcam, Cambridge, England).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: IL4I1 Is a Novel Regulator of M2 Macrophage Polarization That Can Inhibit T Cell Activation via L-Tryptophan and Arginine Depletion and IL-10 Production

doi: 10.1371/journal.pone.0142979

Figure Lengend Snippet: BMDMs were transfected with siRNA to target IL4I1 or with scrambled siRNA for 24 h. The silencing efficiency was evaluated by q-PCR (A) and western blotting (B), respectively. Representative data are presented as means ± S.D. of three independent experiments. Significance was calculated by one-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate significant differences compared to scrambled siRNA transfections; ***p<0.001. BMDMs were transfected with siRNA against IL4I1 or scrambled siRNA for 24 h, and then were treated with IL-4 (20 ng/mL) or were untreated for 24 h. Expression of Arg-1 and IL4I1 was assayed by western blotting (C) and IL-10 expression was assayed by ELISA (F). The gene expression levels of the M2 markers Fizz-1, Arg-1, YM-1, and MR were assayed by q-PCR (E). BMDMs were infected with IL4I1-encoding recombinant retrovirus or control, then were treated with IL-4 (20 ng/mL) or were untreated for 24 h. Levels of gene expression for the M2 markers Fizz-1, Arg-1, YM-1, and MR were assayed by q-PCR (D) and IL-10 expression was assayed by ELISA (F). Representative data are presented as means ± S.D. of four independent experiments. Significance was calculated by one-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate statistically significant differences, either compared to controls, or between two conditions that are linked by a bar (*p<0.05, **p<0.01, ***p<0.001). BMDMs were stimulated with IL-4 (20 ng/mL) or left untreated for 5, 30, or 120 min. The phosphorylation state of STAT-3 and STAT-6 was assessed by western blotting (G); blots are representative of four experiments. Total STAT-6, total STAT-3, and GADPH were used as loading controls.

Article Snippet: Rabbit monoclonal anti-Myc epitope-tagged antibody, and phospho-STAT6 (Tyr641), phospho-STAT3 (Tyr705), total STAT-3, and total STAT-6 monoclonal antibodies were from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Infection, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dengue virus type 2 antagonizes IFN-alpha but not IFN-gamma antiviral effect via down-regulating Tyk2-STAT signaling in the human dendritic cell.

doi: 10.4049/jimmunol.174.12.8163

Figure Lengend Snippet: FIGURE 3. Tyrosine-phosphorylation of STATs in DV-infected DCs. Human DCs at 1 106/ml (4–7 106 cells in average in each treatment condition) in- fected by mock or DV (NGC strain) at MOI 5 at various time points were collected and lysed, and the whole ly- sates were prepared. Aside from the 3-h time point that was within the period of viral absorption, the rest of the time points shown all included the first 4 h for viral absorption. Western blotting was performed to deter- mine the levels of total (-t) and tyrosine-phosphorylated (-p) STAT1 and STAT2 (A), STAT3 (B), STAT5 and STAT6 (C) as described in Materials and Methods. As a positive control, IFN- (1,000 U/ml) was added 5 min before cell collection. The data shown are representative of three to six independent experiments using different donor DCs with similar results.

Article Snippet: Abs against total STAT1, STAT2, STAT3, STAT5, and STAT6 were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Infection, Western Blot, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dengue virus type 2 antagonizes IFN-alpha but not IFN-gamma antiviral effect via down-regulating Tyk2-STAT signaling in the human dendritic cell.

doi: 10.4049/jimmunol.174.12.8163

Figure Lengend Snippet: FIGURE 4. DV induced DNA-binding activities of STATs in DCs. To determine the DNA-binding activi- ties of STATs, human DCs 1 106/ml (1–2 107 cells in average in each treatment condition) were infected with mock or DV (NGC strain) for various periods of time, and the cells were collected and nuclear extracts were prepared. The DNA-binding activities of STAT3 (A) or STAT1, STAT5, and STAT6 (B) in nuclear ex- tracts of different treatment conditions were analyzed by EMSA as described in Materials and Methods. The fold induction was presented as a comparison with the signal intensity in mock-infected control. The results were ob- tained from at least three to six different donors DC with similar results.

Article Snippet: Abs against total STAT1, STAT2, STAT3, STAT5, and STAT6 were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Infection, Comparison, Control