Journal: Scientific reports

Article Title: Caerin 1.1/1.9-mediated antitumor immunity depends on IFNAR-Stat1 signalling of tumour infiltrating macrophage by autocrine IFNα and is enhanced by CD47 blockade.

doi: 10.1038/s41598-025-87687-0

Figure Lengend Snippet: Fig. 4. The anti-tumor effect of F1/F3 relies on the STAT1 related axis. (A) Experimental timeline and design for F1/F3 treatment. (B) Levels of p-STAT1 (Y701) in DCs (CD45.2+CD11c+MHCII+) and macrophages (CD45.2+CD11b+F4/80+) were assessed following F1/F3, control P3 peptide, or saline treatment, using intracellular staining (ICS) and flow cytometry. (C) Survival rates and tumour volumes in treated and control mice administered 2 mg fludarabine or 100 µl PBS intraperitoneally every other day. (E) Survival analysis of mice with STAT1 inhibition in conjunction with F1/F3 treatment. Each experiment included 4–10 mice per group, with data representative of one independent experiment, shown as mean ± SD or mean ± SEM (D). *P < 0.05; **P < 0.01; ***P < 0.001; by one-way ANOVA test (B) or simple survival analysis (Kaplan–Meier) (C,E) or two-way ANOVA test (D).

Article Snippet: Antibodies against p-Stat1 (Y701) (clone: D4A7), p-Stat1 (S727) (clone: 8826), β-actin were used in this study, all of which were purchased from Cell Signalling Technology.

Techniques: Control, Saline, Staining, Flow Cytometry, Inhibition

Journal: Scientific reports

Article Title: Caerin 1.1/1.9-mediated antitumor immunity depends on IFNAR-Stat1 signalling of tumour infiltrating macrophage by autocrine IFNα and is enhanced by CD47 blockade.

doi: 10.1038/s41598-025-87687-0

Figure Lengend Snippet: Fig. 7. Schematic diagram of mechanisms activated by caerin peptide therapy. Following Caerin peptide administration, there is an increase in immune cell infiltration into the tumour, including T cells, macrophages, and CD45.2+ cells. Activated macrophages secrete IFNα, which initiates the IFNAR-1/STAT1 signalling pathway. This pathway boosts macrophage sensitivity to type I IFN, enhancing their function. Consequently, macrophages polarise into an inflammatory phenotype with potent anti-tumour activity, contributing to effective tumour treatment.

Article Snippet: Antibodies against p-Stat1 (Y701) (clone: D4A7), p-Stat1 (S727) (clone: 8826), β-actin were used in this study, all of which were purchased from Cell Signalling Technology.

Techniques: Activity Assay

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 2 CEP-33779 concentration-dependently suppressed the activation of the JAK2-STAT1 pathway and upregulation of PD-L1 on freshly isolated AMs in an LPS-induced ALI mouse model as shown by immunofluorescence staining. Cells in BALF were separated from each group of mice for immunofluorescence staining, and the expression of p-JAK2, p-STAT1, and PD-L1 was visualized using laser confocal microscopy. Blue fluorescence corresponds to DAPI-stained nuclei; Red fluorescence represents CD11c stained with CD11c antibodies, marking alveolar macrophages. Green fluorescence indicates staining for phosphorylated JAK2 antibodies, phosphorylated STAT1 antibody and PD-L1 antibodies respectively. All laser confocal microscopy photos were captured using a 40x oil mirror. Scale bars indicate 20 mm.

Article Snippet: Cells were then incubated overnight at the indicated dilution in Immunol Staining Primary Antibody Dilution Buffer (Beyotime, cat#: P0103) containing rabbit anti-p-JAK2 antibody (diluted 1:1000; Abcam, cat#: ab32101), rabbit anti-p-STAT1 antibody (diluted 1:100; Cell Signaling Technology, cat#: 9177), mouse anti-PD-L1/CD274 antibody(diluted 1:100; Proteintech, cat#: 66248-11-Ig), and rabbit anti-CD11c antibody(diluted l: l00; Signalway Antibody,cat#: C91504FITC).

Techniques: Concentration Assay, Activation Assay, Isolation, Staining, Expressing, Confocal Microscopy

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 3 The activation of the JAK2-STAT1 signaling pathway and PD-L1 expression in MH-S cells was induced by LPS. MH-S cells were stimulated with LPS (1 mg/ml) for different durations: 0, 15, 30, 60, and 120 minutes. Western blot analysis was performed to detect the phosphorylation levels and total protein levels of JAK2 and STAT1. A significant increase in phosphorylation of JAK2 and STAT1 was observed at 15 minutes after LPS stimulation (A, B). PD-L1 expression was enhanced after LPS stimulation for 24 and 48 hours (C). Quantitative analysis of the intensity of pJAK2, pSTAT1, and PD-L1 bands (D–F). ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Cells were then incubated overnight at the indicated dilution in Immunol Staining Primary Antibody Dilution Buffer (Beyotime, cat#: P0103) containing rabbit anti-p-JAK2 antibody (diluted 1:1000; Abcam, cat#: ab32101), rabbit anti-p-STAT1 antibody (diluted 1:100; Cell Signaling Technology, cat#: 9177), mouse anti-PD-L1/CD274 antibody(diluted 1:100; Proteintech, cat#: 66248-11-Ig), and rabbit anti-CD11c antibody(diluted l: l00; Signalway Antibody,cat#: C91504FITC).

Techniques: Activation Assay, Expressing, Western Blot, Phospho-proteomics

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 4 The effect of CEP-33779 and Fludarabine on p-JAK2, p-STAT1, and PD-L1 expression in MH-S cells was analyzed using western blot and immunofluorescence. MH-S cells were pre-treated with varying concentrations (10, 100, 1000, and 3000 nM) of CEP-33779 for one hour before stimulation with 1 mg/ml of LPS for two hours. Subsequently, western blot analysis was performed to detect the phosphorylation levels and total protein levels of JAK2 (A) and STAT1 (B). Quantitative analysis was conducted to determine the levels of phosphorylated JAK2, phosphorylated STAT1 and PD-L1 expression (C, D, I, J, L). Representative confocal microscopy images from three independent experiments are shown in panels (E–G). MH-S cells in the LPS group were stimulated with 1 mg/ml LPS for either 2 hours (for p-JAK2 and p-STAT1 expression) or 24 hours (for PD-L1 expression). In the LPS+CEP-33779 group, MH-S cells were pretreated with 3000 nM CEP-33779 for either 1 hour or 2 hours, followed by co- treatment with 1 mg/ml LPS and 3000 nM CEP-33779 for either 2 or 24 hours. Blue fluorescence indicates cell nuclei stained with DAPI; (E, F) show green fluorescence representing p-JAK2 and p-STAT1 with nuclear translocation expression respectively; (G) shows red fluorescence indicating PD- L1. All laser confocal microscopy photos were taken using a 40x oil mirror. (H, K) cells are pretreated with 50 mM of the STAT1 inhibitor fludarabine (MCE, cat number: 21679-14-1) or without it for 24 h. Subsequently, cells are treated with 1 mg/ml LPS for 30 min to detect the levels of p-JAK2 and p-STAT1. In addition, the protein expression levels of PD-L1 are detected by pretreatment with or without 50 mM fludarabine for 24 h followed by treatment with 1 mg/ml LPS for 48 h. The scale bar is 20 mm. The results are presented as Mean ± SE from three independent experiments (ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Article Snippet: Cells were then incubated overnight at the indicated dilution in Immunol Staining Primary Antibody Dilution Buffer (Beyotime, cat#: P0103) containing rabbit anti-p-JAK2 antibody (diluted 1:1000; Abcam, cat#: ab32101), rabbit anti-p-STAT1 antibody (diluted 1:100; Cell Signaling Technology, cat#: 9177), mouse anti-PD-L1/CD274 antibody(diluted 1:100; Proteintech, cat#: 66248-11-Ig), and rabbit anti-CD11c antibody(diluted l: l00; Signalway Antibody,cat#: C91504FITC).

Techniques: Expressing, Western Blot, Phospho-proteomics, Confocal Microscopy, Staining, Translocation Assay

Journal: Cells

Article Title: Threonine Phosphorylation and the Yin and Yang of STAT1: Phosphorylation-Dependent Spectrum of STAT1 Functionality in Inflammatory Contexts

doi: 10.3390/cells13181531

Figure Lengend Snippet: Immunoblotting antibodies list.

Article Snippet: P-Stat1 (S727) (D3B7) rabbit mAb , CST , 8826S.

Techniques: Western Blot

Journal: Cells

Article Title: Threonine Phosphorylation and the Yin and Yang of STAT1: Phosphorylation-Dependent Spectrum of STAT1 Functionality in Inflammatory Contexts

doi: 10.3390/cells13181531

Figure Lengend Snippet: Thr748 phosphorylation is dispensable for Stat1-mediated cytokines expression and IFN signaling following pristane-induced lupus. ( A – G ) Splenocytes from pristane-injected Wt and T748A littermates. Total RNA was isolated, and the indicated transcripts were quantified by qRT PCR. Data are presented as medians ( n = 3 biological replicates). ( H ) Splenocytes from naïve and pristane-injected Wt and T748A littermates. Whole-cell lysates were harvested and separated by SDS PAGE. The indicated endogenous proteins were detected by Western blotting analysis. Data show three independent biological replicates per genotype per group. ( I ) Quantification of mean band intensities of ( H ). p values are shown as measured by unpaired student’s t test with Welch’s correction ( A – G ) and one-way ANOVA with post hoc Tukey’s test ( I ). NT, non-treated.

Article Snippet: P-Stat1 (S727) (D3B7) rabbit mAb , CST , 8826S.

Techniques: Phospho-proteomics, Expressing, Injection, Isolation, Quantitative RT-PCR, SDS Page, Western Blot

Journal: Cells

Article Title: Threonine Phosphorylation and the Yin and Yang of STAT1: Phosphorylation-Dependent Spectrum of STAT1 Functionality in Inflammatory Contexts

doi: 10.3390/cells13181531

Figure Lengend Snippet: Thr748 phosphorylation is dispensable for STAT1-mediated pathology in pristane-induced lupus. ( A ) Survival rate of pristane-injected KO and T748A littermates. ( B , C ) Serum levels of anti-dsDNA IgG of naïve and pristane-injected KO and T748A littermates as measured by ELISA. ( D ) Measurements of the levels of albumin/creatinine in urine of pristane-injected KO and T748A littermates as measured by ELISA. ( E ) Histopathological analysis of glomerulonephritis. Data are representative of two independent experiments. n = 4–8 mice per genotype per group for each experiment. Scale bar, 100 μm. p values are shown as measured by log-rank test (Mantel-Cox) ( A ), one-way ANOVA with post hoc Tukey’s test ( B ), or unpaired student’s t test with Welch’s correction ( C , D ). NT, non-treated.

Article Snippet: P-Stat1 (S727) (D3B7) rabbit mAb , CST , 8826S.

Techniques: Phospho-proteomics, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Threonine Phosphorylation and the Yin and Yang of STAT1: Phosphorylation-Dependent Spectrum of STAT1 Functionality in Inflammatory Contexts

doi: 10.3390/cells13181531

Figure Lengend Snippet: Scheme for a phosphorylation-dependent spectrum of STAT1 functionality in inflammatory contexts: IFN phospho-tyrosine-dependent and inflammatory phospho-threonine-dependent.

Article Snippet: P-Stat1 (S727) (D3B7) rabbit mAb , CST , 8826S.

Techniques: Phospho-proteomics

Journal: Toxicologic Pathology

Article Title: Biocompatible Solutions: Evaluating the Safety of Repeated Intra-Articular Injections of pMPCylated Liposomes for Knee Osteoarthritis Therapy in Rat Models

doi: 10.1177/01926233241271400

Figure Lengend Snippet: Antibodies used for the immunofluorescence study.

Article Snippet: P-Stat1 , Cell Signaling , 8826 , M1-specific macrophage marker , Green nuclear signal.

Techniques: Immunofluorescence, Marker

Journal: Toxicologic Pathology

Article Title: Biocompatible Solutions: Evaluating the Safety of Repeated Intra-Articular Injections of pMPCylated Liposomes for Knee Osteoarthritis Therapy in Rat Models

doi: 10.1177/01926233241271400

Figure Lengend Snippet: Immunofluorescence images of the knee joint in study 1. (A-D) These immunofluorescence (IF) images capture the right knee joint of an animal from Group 3M, which received three repeated IA injections of 15 mM CCoat. Positive cells for CD68 and CD163 cocktail are indicated by red cytoplasm, C-Maf signal is indicated by pink/purple nucleus, Pstat1 by green nuclear signal and DAPI by the blue nucleus. Autofluorescence is present within the tibia and femur, which presents as yellow color. A. The white arrows highlight the synovial membranes with IF-positive macrophages. B. IF-positive cells expressing CD68 and CD163 within the synovial membrane are observed in this image. The red cytoplasm is indicated by white arrows, while the nucleus, highlighted in blue and positive for DAPI, is marked by yellow arrows. C. These are the same cells as depicted in Figure B. The C-Maf signal is represented by the pink/purple coloration in the nucleus (white arrows), signifying the presence of M2 macrophages. D. These cells correspond to those shown in Figure B. The green nuclear signal, denoting positive P-Stat1 binding, is marked by white arrows. However, it is noteworthy that the signal is diminished both in area and intensity. (E-H) These IF images capture the right knee joint of an animal from Group 4F, which received three repeated IA injections of 30 mM CCoat. Positive cells for CD68 and CD163 cocktail are indicated by red cytoplasm, C-Maf signal is indicated by pink/purple nucleus, Pstat1 signal by green nuclear signal and DAPI by the blue nucleus. E. The white arrows point to synovial membranes with immunofluorescence-positive macrophages. F. Cells within the synovial membrane exhibit positive IF for CD68 and CD163. The red cytoplasm is indicated by white arrows, while the nucleus, highlighted in blue (yellow arrows), confirms DAPI positivity. G. These cells correspond to those in Figure F. The C-Maf signal is represented by the pink/purple coloration in the nucleus (white arrows), indicating the presence of M2 macrophages. H. These are the same cells as shown in Figure F. The green nuclear signal, indicative of positive P-Stat1 binding, is observed, but notably reduced in both area and intensity.

Article Snippet: P-Stat1 , Cell Signaling , 8826 , M1-specific macrophage marker , Green nuclear signal.

Techniques: Immunofluorescence, Expressing, Membrane, Binding Assay