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Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, <t>STAT1,</t> STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001
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Modulation of intracellular signaling pathways in HD, RA, and SLE cells exposed to environmental contaminants. Heatmap showing the relative expression and phosphorylation levels of key signaling proteins involved in immune regulation, inflammation, and cell survival (STAT1, STAT3, p38, AKT, and NFκB) and their phosphorylated forms in HD, RA, and SLE after exposure to PM, silica, and TCDD. Data are represented as z-score normalized values. Asterisks indicate statistically significant differences compared with the unstimulated control within each group (p < 0.05). AKT: protein kinase B; HD: healthy donors; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; P: phosphorylated form; p38: p38 mitogen-activated protein kinase; PM: particulate matter; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; STAT: signal transducer and activator of transcription; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Journal: Journal of Translational Autoimmunity

Article Title: Exploring the immunomodulatory effects of environmental contaminants on autoimmune patients: An in vitro approach

doi: 10.1016/j.jtauto.2025.100341

Figure Lengend Snippet: Modulation of intracellular signaling pathways in HD, RA, and SLE cells exposed to environmental contaminants. Heatmap showing the relative expression and phosphorylation levels of key signaling proteins involved in immune regulation, inflammation, and cell survival (STAT1, STAT3, p38, AKT, and NFκB) and their phosphorylated forms in HD, RA, and SLE after exposure to PM, silica, and TCDD. Data are represented as z-score normalized values. Asterisks indicate statistically significant differences compared with the unstimulated control within each group (p < 0.05). AKT: protein kinase B; HD: healthy donors; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; P: phosphorylated form; p38: p38 mitogen-activated protein kinase; PM: particulate matter; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; STAT: signal transducer and activator of transcription; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBS-T and incubated overnight at 4 °C with primary antibodies against phospho-AKT (Thr308), phospho-NFκB p65 (Ser536), phospho-p38 MAPK (Thr180/Tyr182), phospho-STAT1 (Tyr701), and phospho-STAT3 (Tyr705) (Cell Signaling Technology, Danvers, MA, USA; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Control

Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software