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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Curcumin, a natural polyphenol, has been reported to modulate various oncogenic signaling pathways, but its role in inhibiting IFN-γ-driven PD-L1 expression in NSCLC remains unclear.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The NSCLC cell line A549 were treated with curcumin (50 µM) for 2 h before stimulation with IFN-γ (500 U/ml).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Western blot, qRT-PCR, and immunofluorescence microscopy were used to evaluate STAT1 phosphorylation, PD-L1 expression, and the localization of phosphorylated STAT1 (p-STAT1).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The expression of interferon-stimulated genes (ISGs), including SOCS1 and ISG15, was also examined.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Additionally, the Resazurin assay was performed to assess cell viability.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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IFN-γ significantly induced STAT1 phosphorylation, leading to a time-dependent upregulation of PD-L1 expression.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Immunofluorescence confirmed that p-STAT1 is translocated to nucleus.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Curcumin treatment inhibited STAT1 phosphorylation by 68% (p < 0.001), leading to a marked reduction in PD-L1 expression.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Moreover, curcumin suppressed IFN-γ-induced SOCS1 (63%) and ISG15 (54%) expressions, indicating a broader effect on STAT1-mediated immune evasion.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Finally, curcumin enhanced IFN-γ-mediated growth inhibition, reducing cell viability by 47% at 48 h (p < 0.01).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Curcumin effectively inhibits IFN-γ-induced STAT1 phosphorylation and PD-L1 expression, downregulates ISGs, and enhances IFN-γ-mediated tumor suppression.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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These findings suggest that curcumin may serve as a therapeutic adjuvant in NSCLC, potentially improving immune checkpoint inhibitor (ICI) efficacy.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Lung cancer continues to be the most prevalent cancer-related mortality globally, responsible for more than 1.8 million deaths per year (Siegel et al. 2024).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Non-small cell lung cancer (NSCLC) accounts for ~ 85% of all lung cancer cases, an issue that is of significant public health importance and also a target for oncological research (Salih et al. 2025).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Over the last decade, the development of immune checkpoint inhibitors (ICIs) targeting the programmed death- 1/programmed death-ligand 1 (PD- 1/PD-L1) axis has shifted the treatment landscape for NSCLC, significantly improving survival rates in a particular group of patients (Rizvi et al. 2015).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Nevertheless, with these advancements, a large proportion of NSCLC patients either show a lack of responsiveness to ICIs (primary resistance) or develop resistance ("acquired resistance") over time that significantly restricts their clinical efficacy (Sharma et al. 2017; Rotte 2019).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Immune evasion of NSCLC is significantly attributed to the expression of PD-L1, a transmembrane molecule that binds to PD- 1 receptors located on cytotoxic T cells and induces immune suppression and tumor tolerance.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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PD-L1 overexpression is often linked to poor prognosis, tumor aggression, and poor response to ICIs (Rizvi et al. 2015; Kim and Chen 2016).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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One of the major regulators of PD-L1 expression in the tumor microenvironment is IFN-γ, a key cytokine modulating the immune system.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Although IFN-γ is classically described to have tumor-suppressive effects, paradoxically, it also plays a pro-tumorigenic role by inducing PD-L1 expression, enabling tumor cells to escape immune surveillance (Garcia-Diaz et al. 2017).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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IFN-γ signaling is largely achieved via Janus Kinase—Signal Transducer and Activator of Transcription (JAK-STAT) pathway and, in particular, via STAT1.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Binding of IFN-γ to its receptors, JAK1 and JAK2, phosphorylates STAT1 on Tyr701.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Upon phosphorylation, STAT1 dimerizes, translocate to the nucleus, and binds to gamma-activated sequence (GAS) motifs of the PD-L1 promoter, in turn increasing the level of PD-L1 transcription (Sumitomo et al. 2022).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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As confirmed by several studies, IFN-γ-induced activation of STAT1 is one of the most potent inducers of PD-L1 expression in NSCLC, which in turn leads to adaptive immune resistance that prevents T-cell-mediated tumor destruction (Drake et al. 2006; Shin et al. 2017).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Since IFN-γ levels are typically increased in the tumor microenvironment of NSCLC, strategies that can inhibit IFN-γ-driven upregulation of PD-L1 promise to be valuable for improving the efficacy of ICIs (Spranger et al. 2013).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Despite extensive research regarding PD-L1 regulation in NSCLC, therapeutic approaches that specifically aim to silence IFN-γ-induced PD-L1 expression are still limited.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Strategies presently are based on ICIs in synergy with kinase inhibitors, epigenetic modulators, or chemotherapy, but such pharmacologic efforts are typically associated with toxicity and short-term failure (Zhou and Yang 2023).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Accordingly, the discovery of non-toxic, naturally derived compounds that effectively and specifically suppress IFN-γ-mediated PD-L1 expression but do not negatively affect the host immune function is of significant interest.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Curcumin, a polyphenolic compound isolated from the extract of Curcuma longa (turmeric), has attracted attention as a multi-targeted therapeutic agent due to its anti-inflammatory, antioxidant, and anti-cancer functions (Allegra et al. 2017).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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It has been shown to suppress a variety of oncogenic signaling pathways such as nuclear factor-kappa B (NF-κB), cyclooxygenase- 2 (COX- 2), STAT3, and AKT, leading to growth inhibition and apoptosis in a variety of cancers (Kumar et al. 2021).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Several preclinical studies have suggested that curcumin can downregulate PD-L1 expression in different cancer models, including breast and colon cancer, by inhibiting STAT1 phosphorylation and preventing its nuclear translocation (Midura-Kiela et al. 2012).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Nevertheless, its specific involvement in downregulating IFN-γ-induced PD-L1 expression in NSCLC, especially in A549 cells, is unknown.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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As high PD-L1 expression is associated with poor response to ICIs, understanding natural products (like curcumin) that could regulate IFN-γ-induced PD-L1 expression might provide new strategies for upgrading the outcome of NSCLC treatment.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Although these encouraging results are promising, there are some deficiencies in our knowledge about how curcumin functions in regulating IFN-γ/STAT1 signaling in lung cancer.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Firstly, while previous reports have shown that curcumin can block the phosphorylation of STAT1, they have not specifically evaluated the effect of curcumin on the expression of PD-L1 in NSCLC.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Second, the underlying mechanism of curcumin's inhibitory action on STAT1 is still unknown, especially whether curcumin directly inhibits STAT1 phosphorylation or inhibits its nuclear translocation.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Third, the ability of curcumin to enhance the efficacy of ICIs by decreasing PD-L1 expression has not been extensively studied, and this should be explored.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Given these gaps in knowledge, the present study aims to investigate the effect of IFN-γ on STAT1 phosphorylation and PD-L1 expression in A549 cells and determine whether curcumin can inhibit IFN-γ-induced STAT1 activation and subsequent PD-L1 upregulation.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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In addition, explore the impact of curcumin on IFN-γ-induced expression of interferon-stimulated genes (ISGs), such as SOCS1 and ISG15, which are involved in immune signaling and tumor immune escape, and evaluate whether curcumin enhances the anti-proliferative effects of IFN-γ, suggesting a potential therapeutic benefit beyond immune modulation.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Human non-small cell lung cancer (NSCLC) cell, A549, was obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies), 1% penicillin–streptomycin, and 2 mM L-glutamine.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Cells were cultured in a humidified incubator at 37 °C and 5% CO₂ with regular 2–3-day passaging in a 0.25% trypsin–EDTA suspension.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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All experiments were performed on cells between passage 5 and passage 15, thereby permitting reproducibility.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Recombinant human IFN-γ was purchased from PeproTech (EC Ltd PeproTech, London, UK) and stored in sterile phosphate-buffered saline (PBS) at 10 U/ml stock concentration.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Curcumin ≥ 95% was purchased from Santa Cruz (Heidelberg, Germany) and dissolved in dimethyl sulfoxide (DMSO) (≥ 99.9% purity) to make a 50-mM solution.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The working dilutions of curcumin were freshly prepared in the complete DMEM prior to each experiment.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Cells were pretreated with curcumin (50 µM) for 2 h prior to the IFN-γ (500 U/ml) stimulation for studied time points.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Control groups were treated with vehicle (DMSO < 0.1%).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Cell viability was assessed using the Resazurin assay (Sigma-Aldrich, #R7017 - 1G).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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A549 cells were seeded in 96-well plates at a density of 3 × 10 cells per well (final volume of 100 μL/well) and allowed to adhere overnight.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Following 24 h treatment with IFN-γ, curcumin, or a combination of both, 20 µl of Resazurin reagent (0.15 mg/ml) was then added to each well, and incubation was performed for 2–4 h at 37 °C.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The conversion of Resazurin to resorufin was measured by fluorescence using a microplate reader (excitation 560 nm, emission 590 nm) or by absorbance at 570 nm with 600 nm as a reference wavelength.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Cell viability was determined as a percentage compared to untreated control cells.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Cell Viability (%) = (Absorbance of control cells of treated cells) × 100.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Total protein lysates were extracted from treated A549 cells using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors and centrifuged at 10,000 × g for 10 min at 4◦C. The total protein concentrations were measured with the bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Equal amounts of protein (20 μg per sample) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels (SDS-PAGE) and transferred onto 0.45 μM nitrocellulose membranes (Merck).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween- 20 (TBST) for 1 h at room temperature to prevent non-specific binding and incubated overnight at 4 °C with primary antibodies against phospho-STAT1 (Tyr701), total STAT1, PD-L1, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse IgG antibody or anti-rabbit IgG, HRP-linked antibody, for 1 h at room temperature.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Protein bands were detected using enhanced chemiluminescence (ECL) reagent (GE Healthcare, USA) and visualized using a ChemiDoc imaging system (Bio-Rad, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Analysis of protein bands was performed using ImageJ software (NIH, Bethesda, MD, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The primary and secondary antibodies are listed in Table 1.Table 1The description of primary and secondary antibodiesAntibodySpeciesCloneDilutionCa#SourcePD-L1RabbitMonoclonal1:1000ab213524AbcamSTAT1MouseMonoclonal1:10009176Cell Signaling TechnologypSTAT1RabbitMonoclonal1:10009177SCell Signaling Technologyβ-actinMouseMonoclonal1:500047,778Santa Cruz Biotechnology, Dallas, TX, USAAnti-mouseHorse-1:10,0007076Cell Signaling, Danvers, MA, USAAnti-RabbitGoat-1:10,0007074Cell Signaling, Danvers, MA, USA The description of primary and secondary antibodies Total RNA was extracted from A549 cells using the RNeasy Mini-Kit (Qiagen, #74,104) according to the manufacturer’s instructions, and the eluted RNA purity and concentration were assessed using a NanoDrop One spectrophotometer (Thermo Scientific, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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For cDNA synthesis, 500 ng of RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit to cDNA as per the manufacturer’s instructions.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Quantitative real-time PCR (qRT-PCR) was conducted using the SYBR Green PCR Master Mix (Applied Biosystems™) on a QuantStudio 5 Machine (Thermo Fisher Scientific, Inc.).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The relative expression levels of PD-L1, STAT1, and ISGs (ISG15 and SOCS1) were normalized to the housekeeping gene GAPDH and analyzed using the 2^ − ΔΔCt method.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The PCR reactions were carried out in duplicate with 40 cycles of denaturation (15 s at 95 °C), annealing (20 s at 65 °C), and elongation (20 s at 72 °C) after an initial enzyme activation (15 min at 95 °C).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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The primer sequences used are presented in Table 2.Table 2List of PCR primers designed using NCBI/Primer-BLAST programPrimerPrimer sequencesForwardReverseCD274 ()5′-TGGCATTTGCTGAACGCATTT- 3′5′-AGTGCAGCCAGGTCTAATTGT- 3′ISG155′-ATCACCCAGAAGATCGGCGT- 3′5′-TCGCATTTGTCCACCACCAG- 3′SOCS15’- TTCGCCCTTAGCGTGAAGATGG- 3′5’- TAGTGCTCCAGCAGCTCGAAGA- 3′GAPDH5′-GGAAGGTGAAGGTCGGAGTC- 3′5′-TGAAGGGGTCATTGATGGCA- 3′ List of PCR primers designed using NCBI/Primer-BLAST program A549 cells were seeded onto sterile coverslips in 12-well plates and treated as described.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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After incubation, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X- 100 for 10 min.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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After blocking with 1% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against PD-L1 and phospho-STAT1.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Following PBS washes, cells were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies for 1 h at room temperature.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Nuclei were counterstained with DAPI, and images were captured using a fluorescence microscope (Zeiss Axio Observer, Germany).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Statistical analyses were conducted using GraphPad Prism 10 (GraphPad Software, USA).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare multiple groups, and an unpaired Student’s t-test was used for pairwise comparisons.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Differences were considered statistically significant at p < 0.05.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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STAT1 phosphorylation at tyrosine 701 (Tyr701) is a key regulatory event in IFN-γ-mediated signaling, leading to STAT1 dimerization, nuclear translocation, and transcriptional activation of ISGs, including PD-L1.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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To confirm the activation of the JAK-STAT1 pathway in A549 cells, we analyzed phospho-STAT1 (Tyr701) levels by Western blotting after stimulation with 500 U/ml of IFN-γ for different time points (0, 1, 2, 6, 12, and 24 h).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Our results demonstrated a time-dependent increase in STAT1 phosphorylation.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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A significant induction observed as early as 30 min post-treatment (p < 0.01), peaking at 2 h (p < 0.001), and remaining elevated up to 24 h. Total STAT1 protein levels remained unchanged across all time points, indicating that the increase in phospho-STAT1 was due to phosphorylation rather than upregulation of STAT1 expression, as shown in Fig. 1.Fig.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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1Western blot analysis showing STAT1 phosphorylation and PD-L1 expression in A549 NSCLC cells following IFN-γ treatment.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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A549 cells were treated with 500 U/mL of IFN-γ for various time points (0, 30 min, 1 h, 2 h, and 24 h).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Protein lysates were collected and analyzed by Western blot using antibodies against phosphorylated STAT1 (Tyr701), total STAT1, and PD-L1.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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β-Actin was used as a loading control Western blot analysis showing STAT1 phosphorylation and PD-L1 expression in A549 NSCLC cells following IFN-γ treatment.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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A549 cells were treated with 500 U/mL of IFN-γ for various time points (0, 30 min, 1 h, 2 h, and 24 h).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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Protein lysates were collected and analyzed by Western blot using antibodies against phosphorylated STAT1 (Tyr701), total STAT1, and PD-L1.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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β-Actin was used as a loading control To further investigate the effect of IFN-γ on STAT1 activation, we performed immunofluorescence microscopy to visualize the cellular localization of phosphorylated STAT1 (p-STAT1) in A549 cells following a 2-h treatment with IFN-γ (500 U/ml).
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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In untreated control cells, STAT1 was primarily localized in the cytoplasm, with minimal nuclear fluorescence detected.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
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However, upon IFN-γ stimulation, a marked increase in nuclear accumulation of p-STAT1 was observed, as shown in Fig. 2, indicating its activation and translocation to the nucleus, where it functions as a transcription factor.
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PMC12141184
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Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
This translocation pattern was confirmed through co-staining with DAPI, a nuclear marker, which showed strong co-localization of p-STAT1 within the nucleus.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
Fig. 2Immunofluorescence microscopy showing p-STAT1 localization in A549 cells treated with IFN-γ.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
A549 cells were treated with 500 U/mL of IFN-γ for 2 h, then analyzed by immunofluorescence microscopy to assess p-STAT1 localization.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
In untreated cells, p-STAT1 was primarily detected in the cytoplasm.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
Following IFN-γ treatment, increased nuclear localization of p-STAT1 was observed.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
DAPI was used for nuclear staining, and β-actin staining was used to visualize the cytoskeleton Immunofluorescence microscopy showing p-STAT1 localization in A549 cells treated with IFN-γ.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
A549 cells were treated with 500 U/mL of IFN-γ for 2 h, then analyzed by immunofluorescence microscopy to assess p-STAT1 localization.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
In untreated cells, p-STAT1 was primarily detected in the cytoplasm.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
Following IFN-γ treatment, increased nuclear localization of p-STAT1 was observed.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
DAPI was used for nuclear staining, and β-actin staining was used to visualize the cytoskeleton Studies reveal that IFN-γ dramatically increases the expression of PD-L1 on cancer cells, mainly by activating the STAT1 signaling pathway.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
To validate this association, we examined PD-L1 upregulation upon IFN-γ treatment in A549 cells.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
Western blot analysis revealed a time-dependent increase in PD-L1 protein levels, with minimal expression in untreated control cells.
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
Upon IFN-γ stimulation, PD-L1 levels began to increase at 30 min and 1 h, corresponding with the early activation of STAT1 phosphorylation (p-STAT1, Tyr701).
|
PMC12141184
|
Curcumin inhibits IFN-γ induced PD-L1 expression via reduction of STAT1 Phosphorylation in A549 non-small cell lung cancer cells.
|
By 2 h, PD-L1 expression was noticeably upregulated, and by 24 h, it reached its maximum induction (p < 0.01).
|
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