il 25  (R&D Systems)

Journal: Respiratory Research

Article Title: Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts

doi: 10.1186/s12931-025-03479-0

Figure Lengend Snippet: Transcriptional regulation of SLC40A1 by TGF-β1. a HPFs were treated for 48 h with different cytokines [TGF-β1 (5 ng/ml)​​, WNT-1(50 ng/ml), WNT-3a (50 ng/ml), WNT-5a (50 ng/ml), TNFα (10 ng/ml)​, LPS (1,000 ng/ml), IL-1β (10 ng/ml), IL-4 (20 ng/ml), IL-5 (20 ng/ml), IL-13 (25 ng/ml), IL-21 (25 ng/ml), IL-25 (25 ng/ml), IL-33 (30 ng/ml), IFNβ (1 ng/mL), or IFNγ (20 ng/ml)]. SLC40A1 mRNA levels were determined by real-time PCR and expressed a ratio of the control (CON). b HPFs were treated with 50 ng/ml IL-6 for 48 h or serum-starved for 4 h or 24 h and then treated with 10 nM thrombin or 100 nM factor Xa for 48 h and SLC40A1 mRNA levels were determined by real-time PCR. c HPFs were co-transfected with the pGL3-Basic vector containing the SLC40A1 promoter fragment and firefly luciferase gene (180 ng) along with pRL-TK plasmid containing Renilla luciferase gene (20 ng) and then treated with 5 ng/ml TGF-β1 for 48 h. Firefly and Renilla luciferase activities in cell lysates were measured. The relative firefly luciferase activities were normalized to the respective Renilla luciferase activities. The results are presented as a ratio CON. d HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h. SMAD2 and SMAD3 protein levels were determined by western blot and quantitated. e HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h and then treated with 5 ng/ml TGF-β1 for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. f HPFs were treated with SIS3 (10 µM, Milipore Sigma, Cat# 566405) for 2 h and then with TGF-β1 (5 ng/ml) for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. g HPFs were treated with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to chromatin immunoprecipitation (ChIP) using anti-SMAD3 antibodies. The SMAD3 binding site on the SLC40A1 promoter was detected by real-time PCR. n =3, * p < 0.05, ** p < 0.01, *** p < 0.001 v.s CON or shCON. ## p < 0.01 vs ShCon/TGF-β1. Student t-test ( c ), one-way ANOVA ( a and d ) or two-way ANOVA ( b , e , f and g ). followed by uncorrected Fisher’s least significant difference (LSD) test ( e , f and g ) or Turkey’s multiple comparison test ( b )

Article Snippet: HPFs (1x10 5 /well) were seeded into 12-well plates and treated with different cytokines and other compounds for 48 h: TNF-α (10 ng/ml, #300-01A, PeproTech, Cranbury, NJ, USA), lipopolysaccharides (LPS) (1,000 ng/ml, #00–4976-93, Invitrogen), TGF-β1 (5 ng/ml, #240-B, R&D Systems, Minneapolis, MN, USA) , IL-4 (20 ng/ml, #200-04, PeproTech), IFN-γ (20 ng/ml, #300-02, PeproTech) , IL-21 (25 ng/ml, #200-21, PeproTech), IL-13 (25 ng/ml, #200-13, PeproTech), IFN-β (1 ng/mL, #300-02BC, PeproTech), IL-25 (25 ng/ml, #1258-IL, R&D Systems), IL-33 (30 ng/ml, #200-33, PeproTech), WNT-1 (50 ng/ml, #120-17, PeproTech), IL-1β (10 ng/ml, #200-01B, PeproTech), IL-5 (20 ng/ml, #200-05, PeproTech), WNT-3a (50 ng/mL, #5036-WN, R&D Systems), WNT-5a (50 g/mL, #645-WN, R&D Systems), IL-6 (50 ng/ml, #200-06, Gibco), thrombin (10 nM, #RP-43100, Invitrogen), factor Xa (100 nM, #RP-43114, Invitrogen), ferric ammonium citrate (FAC) (200 μM, # RES20400 -A7, Sigma), deferoxamine (DFO) (10 μM #D9533, Sigma).

Techniques: Real-time Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Luciferase, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 expression positively correlates with proinflammatory markers in M1-polarized macrophages. (A-C) PD-L1-overexpressing (PD-L1 HI ) and control (PD-L1 NC ) THP-1-M cells were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h followed by RNA sequencing. A Heatmap of inflammatory gene expression in PD-L1 HI vs. PD-L1 NC cells. B MA plot showing differential gene expression between PD-L1 HI and PD-L1 NC cells. C TPM expression values of IL-6, IL-27 and NOS2. D - E PD-L1 HI and PD-L1 NC cells were stimulated with LPS + IFN-γ for 24 h. ( n = 3 independent biological replicates) ( D ) qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). E ELISA for IL-6/IL-27 and Griess reagent assay for NO in supernatants ( n = 3 independent biological replicates). (F-G) PD-L1-knockout THP-1-M (PD-L1 KO /THP-1-M) and PD-L1 NC /THP-1-M were stimulated with LPS + IFN-γ for 24 h. F qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). G ELISA and Griess reagent detection of IL-6/IL-27 proteins and NO in supernatants ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C, D, E, F and G was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Expressing, Control, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Triple-verification strategy (gene silencing/antibody neutralization/site-directed mutagenesis) confirms regulatory hierarchy of PD-L1/AIM2/IL-18/STAT1 signaling axis in macrophage M1 polarization. A PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were pretreated with AIM2-targeting siRNA or scramble siRNA (scr siRNA) prior to 24 h LPS (100 ng/ml) + IFN-γ (20 ng/ml) co-stimulation, with subsequent Western blotting analysis of STAT1 phosphorylation. B - C Anti-IL-18 neutralizing antibody (1 µg/ml) and isotype control (1 µg/ml) were pretreated for 24 h followed by LPS + IFN-γ 24 h co-stimulation. B qRT-PCR quantification of IFN-γ mRNA ( n = 3 independent biological replicates). C STAT1 phosphorylation profiling by Western blotting. D Truncation mutant validation: LPS + IFN-γ-stimulated (24 h) PD-L1 Δ35–90 cells analyzed for STAT1 activation vs. full length type. E Following AIM2 silencing in PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells, qRT-PCR quantification of IL-6, IL-27 and NOS2 mRNA after 24 h LPS + IFN-γ stimulation ( n = 3 independent biological replicates). F Post IL-18 neutralization, mRNA levels of IL-6, IL-27 and NOS2 were assessed by qRT-PCR under LPS + IFN-γ 24 h stimulation ( n = 3 independent biological replicates). G STAT1-silenced cells were analyzed for IL-6/IL-27/NOS2 transcriptional changes post 24 h LPS + IFN-γ exposure ( n = 3 independent biological replicates). H Truncated PD-L1 Δ35–90 mutants were evaluated for IL-6, IL-27 and NOS2 mRNA expression following LPS + IFN-γ 24 h challenge ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B, E, F, G and H was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Neutralization, Mutagenesis, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Biomarker Discovery, Activation Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 knockout reduces septic mortality dampens systemic inflammation and alleviates vital organ (cardiac/hepatic/renal) injury in murine models. A PD-L1 wild-type (WT) and knockout (KO) mice were subjected to CLP followed by survival monitoring, inflammatory marker assessment, and multi-organ functional evaluation. B Survival rates of PD-L1 WT and PD-L1 KO mice at indicated time points post-CLP ( n = 15 mice per group). Results were compared by log-rank test. C mRNA levels of IL-6, IL-27, and NOS2 in monocytes analyzed by qRT-PCR at 24 h post-CLP ( n = 3 mice per group). D Serum concentrations of IL-6 and IL-27 proteins measured by ELISA, and NO levels determined by Griess assay at 24 h post-CLP ( n = 3 mice per group). E Serum biomarkers of organ function including ALT and AST for hepatic injury, BUN for renal dysfunction, and cTnI for cardiac damage at 24 h post-CLP ( n = 3 mice per group). The data are presented as the mean ± SEM. Statistical analysis for C, D and E was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Knock-Out, Marker, Functional Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Griess Assay