bioinformatics analysis the myt1l binding sites in the cxcr1 promoter were predicted using zinc finger protein - dna scoring form, a dna binding site predictor for cys2his2 zinc finger proteins ( http: / / zf. princeton. edu / form. php, accessed on 14 march 2025 ). the cancer genome atlas ( tcga ) datasets were used to analyze a relationship between myt1l expression and cxcr1 expression in glioblastoma and neuroblastoma. 4. 12. statistical analysis the student ’ s t - test was used to determine the statistical significance of differences in groα expression, il - 8 expression, cell growth, apoptosis, cell cycle, and luciferase activity between groups. luciferase activity was measured in duplicate, while other experiments were performed in triplic ate. conclusions * * * this work, for the first time, reveals that myt1l functions as a transcription factor governing the expression of cxc chemok ine receptor cxcr1. the cxcr1 signaling promotes proliferation, inhibits apoptosis, and shortens the s phase in dna - pk + glioblastoma cells via activation of the erk1 / 2 pathway, which can be blocked by cxcr1 knockdown. the function of myt1l is dna - pk - dependent, highlighting a key role of dna - pk modulating the transcriptional activity of myt1l. our findings have demonstrated a positive feedback dna - pk / myt1l - cxcr1 - erk1 / 2 proliferative signaling loop in glioblastoma cells and might have significant therapeutic implications. in the future, our studies can be extended by analyzing the effect of cxcr1 knockdown in m059j cells with impaired dna - pk. this would allow us to understand the interlink between dna - pk - myt1l - cxcr1 - perk1 / 2. results ifn - γ significantly induced stat1 phosphorylation, leading to a time - dependent upregulation of pd - l1 expression. immunoflu orescence confirmed that p - stat1 is translocated to nucleus. curcumin treatment inhibited stat1 phosphorylation by 68 % ( p p * * * conclusion * * * curcumin effectively inhibits ifn - γ - induced stat1 phosphorylation and pd - l1 expression, downregulates isgs, and enhances ifn - γ - mediated tumor suppression. these findings suggest that curcumin may serve as a therapeutic adjuvant in nsclc, potentially improving immune checkpoint inhibitor ( ici ) efficacy. material and methods western blot analysis 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 ). equal amounts of protein ( 20 μg per sample ) were separated on 10 % sodium dodecyl sulfate – polyacr ylamide gels ( sds - page ) and transferred onto 0. 45 μm nitrocellulose membranes ( merck ). 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 antibod ies against phospho - stat1 ( tyr701 ), total stat1, pd - l1, and β - actin ( santa cruz biotechnology, dallas, tx, usa ). after washing with tbst, membranes were incubated with horseradish peroxidase ( hrp ) - conjugated secondary anti-mouse igg antibod y or anti-rabbit igg, hrp - linked antibod y, for 1 h at room temperature. protein bands were detected using enhanced chemiluminescence ( ecl ) reagent ( ge healthcare, usa ) and visualized using a chemidoc imaging system ( bio - rad, usa ). analysis of protein bands was performed using imagej software ( nih, bethesda, md, usa ). the primary and secondary antibod ies are listed in table 1. table 1the description of primary and secondary antibod ies antibod yspeciesclonedilutionca # sourcepd - l1rabbitmonoclonal1: 1000ab213524abcamstat1mousemonoclonal1: 10009176cell signaling technologypstat1rabbitmonoclonal1: 10009177scell signaling technologyβ - actinmousemonoclonal1: 500047, 778santa cruz biotechnology, dallas, tx, usa anti-mouse horse - 1: 10, 0007076cell signaling, danvers, ma, usa anti-rabbit goat - 1: 10, 0007074cell signaling, danvers, ma, usa quantitative real - time pcr ( qrt - pcr ) analysis 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 ). 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. 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. ). 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. 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 ). the primer sequences used are presented in table 2. table 2list of pcr primers designed using ncbi / primer - blast program primerprimer sequencesforwardreversecd274 ( pd−l1 ) 5 ′ - tggcatttgctgaacgcattt - 3 ′ 5 ′ - agtgcagccaggtctaattgt - 3 ′ isg155 ′ - atcacccagaagatcggcgt - 3 ′ 5 ′ - tcgcatttgtccaccaccag - 3 ′ socs15 ’ - ttcgcccttagcgtgaagatgg - 3 ′ 5 ’ - tagtgctccagcagctcgaaga - 3 ′ gapdh5 ′ - ggaaggtgaaggtcggagtc - 3 ′ 5 ′ - tgaaggggtcattgatggca - 3 ′ statistical analysis all experiments were performed in triplic ate, and data are presented as mean ± standard deviation ( sd ). statistical analyses were conducted using graphpad prism 10 ( graphpad software, usa ). 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. differences were considered statistically significant at p * * * results * * * discussion immune evasion remains a significant challenge in the treatment of nsclc, with pd - l1 upregulation being one of the primary mechanisms by which tumors escape immune surveillance ( cui et al. 2024 ). the ifn - γ / stat1 signaling pathway plays a crucial role in pd - l1 regulation, enabling tumor cells to suppress t - cell - mediated immune responses and resist immune checkpoint blockade therapy ( padmanabhan et al. 2022 ). in this study, we investigated how ifn - γ - induced stat1 phosphorylation leads to pd - l1 upregulation in a549 cells and explored the potential of curcumin, a bioactive polyphenol with known anti-inflammatory and anti - cancer properties, as a therapeutic agent capable of modulating this pathway. our findings provide strong evidence that curcumin inhibits ifn - γ - induced stat1 activation, thereby reducing pd - l1 expression and enhancing the anti - proliferative effects of ifn - γ in nsclc. consistent with previous studies, we observed that ifn - γ induces a robust phosphorylation of stat1 at tyr701 in a549 cells, leading to its nuclear translocation and subsequent activation of target genes, including pd - l1. the kinetics of stat1 phosphorylation followed a time - dependent pattern, with phosphorylation detected as early as 2 h, peaking at 6 h, and remaining elevated up to 24 h. these results are in agreement with earlier reports that demonstrated a similar pattern of ifn - γ - induced stat1 activation in various cancer models, including melanoma ( schmitt et al. 2012 ), colorectal cancer ( zhao et al. 2020 ), and lung adenocarcinoma ( gao et al. 2018 ). stat1 phosphorylation is a prerequisite for its dimerization and nuclear translocation, which is required for the transcriptional activation of isgs ( wang et al. 2017 ).