Journal: Heliyon

Article Title: Identification of signaling networks associated with lactate modulation of macrophages and dendritic cells

doi: 10.1016/j.heliyon.2025.e42098

Figure Lengend Snippet: Effect of sLA on signaling proteins of interest in DCs and MΦs. ( A ) Schematic experiment was depicted. DCs and MΦs were exposed to either 50 mM sLA or control media, and incubated for 48 h. For Western blot analysis, proteins were separated by size and then transferred to the membrane. Protein detection was done using primary antibodies against several proteins including p-STAT3, p-ERK1/2, p-p38 MAPK, p-STAT1, p-STAT2, p-GSK-3β and β-actin. For flow cytometry, DCs cell suspensions were stained with fluorescently tagged anti-CD11c, p-STAT3, p-ERK and p-p38 MAPK. MΦs were stained with fluorescently tagged anti-F4/80, p-STAT1 and p-GSK-3β. The analysis of the phosphorylated protein expression was performed using a flow cytometer. ( B ), ( D ) Western blot results with represented blots showed the effect of sLA on the levels of p-STAT3, p-ERK1/2 and p-p38 MAPK for DCs. For MΦs, p-STAT1, p-STAT2, p-GSK-3β were shown for the effect of sLA. ( C ), ( E ) Flow cytometric assessment revealed the levels of phosphorylated proteins expression in DCs and MΦs after sLA treatment. The data are demonstrated as the mean ± S.E. from at least three independent experiments, with statistically significant differences calculated by comparing each treatment group to the control. The following symbols indicate the corresponding p-values: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as determined by a two-tailed one-sample t -test.

Article Snippet: The membrane was incubated with each primary antibody targeting phosphorylated STAT1 (p-STAT1, Tyr701, D4A7, Cell signaling technology (CST) #7649), p-STAT2 (Tyr690, CST #4441), p-STAT3 (Ser727, CST #9134), p-SAPK/JNK (Thr183/Tyr185, 81E11, CST #4668), p-SAPK/JNK (Thr183/Tyr185, 98F2, CST #4671), p-NF-κB p65 (Ser536, 93H1, CST #3033), β-Catenin (Ser45, D2U8Y, CST #19807), p-Akt (Ser473, CST #9271), p-Akt (Thr308, D25E6, CST #13038), p-Akt (Ser473, D9E, CST #4060), Akt (pan) (C67E7, CST #4691), p-PTEN (Ser380, CST #9551), p-GSK-3β (Ser9, D85E12, CST #5558), p-c-Raf (Ser259, CST #9421), p-PDK1 (Ser241, C49H2, CST #3438), p-p44/42 MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E, CST #4370), p-p38 MAPK (Thr180/Tyr182, D3F9, CST #4511), and β-actin (CST #4967).

Techniques: Control, Incubation, Western Blot, Membrane, Flow Cytometry, Staining, Expressing, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice

doi: 10.1016/j.jcmgh.2024.01.023

Figure Lengend Snippet: Myeloid deletion of Cdc42 enhances the M2-type differentiation of macrophages via regulating STATs signaling. THP1 cells were pretreated with PMA (100 ng/mL) and ML141 (10 μM) for differentiation and then stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 1 hour for M1 induction, or IL4 and IL13 (both at 40 ng/mL) for 48 hours for M2 induction. The images ( A ) and quantitative results ( C–F ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were detected by Western blot analysis in THP1 cells, n = 3. The images ( B ) and quantitative results ( G–J ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were measured by Western blot analysis in BMDMs stimulated with LPS (100 ng/mL) for 3 hours or IL4 (40 ng/mL) for 48 hours, respectively, n = 3. n. s., no significance; ∗ P < .05; ∗∗∗ P < .001.

Article Snippet: The proteins were extracted and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare), which will be blocked with 5% BSA or skimmed milk in Tris-buffered saline containing 0.1% Tween 20 and probed with antibodies against iNOS (Proteintech, 22226-1-AP), ARG1 (Proteintech, 66129-1-AP), IL10 (proteintech,60269-1-Ig), IL6 (CST, 12912S), SOCS3 (Proteintech, 14025-1-AP), STAT Antibody Sampler Kit (CST, 9939T), Phospho-STAT Antibody Sampler (CST, 9914T: Phospho-STAT1 Tyr701 and Phospho-STAT3 Tyr705, Phospho-STAT6 Tyr641), respectively.

Techniques: Western Blot

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: Nuclear localization of STAT1 and STAT2 is inhibited by HMPV. Human BEAS2b cells were infected with HMPV or mock for 20 h, and treated with IFN for 40 min. After IFN treatment, cells were fixed, stained for immunofluorescence, and images were captured at 40× magnification. The fluorescent intensities of the nuclear and cytosolic STAT1 or STAT2 fluorescence signals was quantified. ( A ) HMPV infection inhibits nuclear translocation of STAT1. ( B ) STAT2 nuclear localization is impaired during HMPV infection. Note reduction of STAT1/2 (red) signal in nuclei of HMPV-infected (green) cells. ** p < 0.01, **** p < 0.0001 compared to Mock + IFN, one-way ANOVA with Tukey post-hoc test. Images are representative of 2 independent experiments. Data in graphs are from two experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Infection, Staining, Immunofluorescence, Fluorescence, Translocation Assay

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV reduces STAT1 and STAT2 activation in primate cells. ( A ) VeroE6 (primate) cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. ( B , C ) Fold change of total STAT1 ( B ) and STAT2 ( C ) expression normalized to value of mock-infected cells. ( D ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells. ( E ) Relative fold change of pSTAT1 and pSTAT2 (fold change of pSTAT1/2 divided by fold change of STAT1/2). * p < 0.05, ** p < 0.01, one sample t test comparison to normalized mock value of 1. Data are representative of ( A ) or combined from ( B – E ) four independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Activation Assay, Infection, Western Blot, Expressing, Comparison

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV infection of murine cells leads to upregulation and phosphorylation of STAT1 and STAT2. ( A ) CMT64/61 (murine), left, and NIH3T3 (murine), right, cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. ( B , C ) Fold change of total STAT1 and STAT2 expression compared to mock-infected wells for CMT64/61 cells. ( D ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected wells in CMT64/61 cells. ( E , F ) For NIH3T3 cells, fold change of STAT1 and STAT2 compared to mock-infected wells. ( G ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells in NIH3T3 cells. * p < 0.05, one sample t test comparing samples to normalized mock value of 1. Data are representative of ( A ) or combined from four ( B – D ) or two ( E – G ) independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Infection, Phospho-proteomics, Western Blot, Expressing

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV does not require STAT1 or STAT2 to inhibit expression and phosphorylation of the other. STAT2-deficient U6A ( A ) and STAT1-deficient U3A ( B ) cells were infected with HMPV for 20 h at an MOI of 1 and 3, treated with IFN for 40 min, and lysed for Western blotting. ( A , B ) Expression and phosphorylation of STAT1 and STAT2 in U6A and U3A cells. ( C ) Fold change of STAT1/pSTAT1 protein levels in U6A cells. ( D ) Fold change of STAT2/pSTAT2 levels in U3A cells. * p < 0.05, ** p < 0.01, one sample t test to normalized mock value of 1. Data are representative of ( A , B ) or combined from ( C , D ) two independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: Expression of human STAT2 promotes STAT1 and STAT2 inhibition, while murine STAT2 inhibits STAT degradation. STAT2-deficient U6A cells were transfected with hSTAT2 or mSTAT2, then infected with HMPV. Cells were treated with IFN 16 h after infection for 40 min before cell lysis and protein harvesting for Western blotting. ( A ) STAT1 and STAT2 expression and phosphorylation in U6A cells in the presence of human or murine STAT2. ( B – E ) Quantification of band intensity as a measure of fold change in U6A cells for total STAT1 ( B ), pSTAT1 ( C ), total STAT2 ( D ), and pSTAT2 ( E ). * p < 0.05, *** p < 0.001, one sample t test against normalized mock value of 1. Data are representative of ( A ) or combined from ( B – E ) two independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Expressing, Inhibition, Transfection, Infection, Lysis, Western Blot, Phospho-proteomics