Journal: Breast Cancer Research : BCR

Article Title: Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E

doi: 10.1186/s13058-025-02193-5

Figure Lengend Snippet: LY6E is identified as a key downstream effector of nuclear PD-L1 in metastasis.​​ A ​​ Left: Venn diagram illustrating the strategy for identifying downstream targets of IFN-γ/nuclear PD-L1 from RNA-seq data of PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. Right: Heatmap of four candidate genes. ​​ B – C ​​ qPCR validation of Cldn1, Ly6e, Anxa8, and Gnb4 mRNA expression in the indicated treatment groups of 4T1 cells ( n = 4). ​​ D ​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. ​​ E – F ​​ qPCR analysis of CLDN1 and LY6E mRNA expression in PD-L1 WT and PD-L1 KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. ​​ G – H ​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. I ​​ Analysis of LY6E and CD274 mRNA expression in TCGA TNBC tumor and adjacent normal tissue samples. ​​ J ​​ Correlation analysis of LY6E and CD274 mRNA expression in TCGA TNBC samples. ​​ K ​​ Representative images of lungs and quantification of metastatic nodules from LY6E overexpression cells. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

Article Snippet: Proteins (20–30 μg) were separated by SDS-PAGE, transferred to nitrocellulose (NC) membranes, and probed with primary antibodies against PD-L1 (CST, #13684; #60475), POLR2A (CST, #2629), LY6E (Proteintech, 22144-1-AP), Claudin-1 (Proteintech, 13050-1-AP), STAT1 and phosphorylated STAT1 (CST, #14994; #9167), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Western Blot, Over Expression

Journal: Breast Cancer Research : BCR

Article Title: Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E

doi: 10.1186/s13058-025-02193-5

Figure Lengend Snippet: Nuclear PD-L1 forms a transcriptional complex with POLR2A to upregulate LY6E.​​ A ​​ Browser view of published ChIP-seq data showing lack of PD-L1 enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​ B ​​ Transcription factors (TFs) predicted to bind the LY6E promoter in MDA-MB-231 cells, ranked by transcriptional potential score. ​​ C ​​ Venn diagram identifying POLR2A as the only TF overlapping between factors predicted to bind LY6E ( B ) and factors found to interact with PD-L1 by Co-IP/MS in MDA-MB-231 cells. ​​ D ​​ Browser view of ChIP-seq data showing POLR2A enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​ E ​​ Predicted structural model of the PD-L1/POLR2A interaction generated by ZDOCK. Key interacting residues are labeled: Salt bridge (PD-L1 R140 - POLR2A E517), Electrostatic interactions (PD-L1 K136/K185 - POLR2A D452/D663). ​​ F ​​ Co-immunoprecipitation assay in MDA-MB-231 cells to detect protein interaction between POLR2A and PD-L1

Article Snippet: Proteins (20–30 μg) were separated by SDS-PAGE, transferred to nitrocellulose (NC) membranes, and probed with primary antibodies against PD-L1 (CST, #13684; #60475), POLR2A (CST, #2629), LY6E (Proteintech, 22144-1-AP), Claudin-1 (Proteintech, 13050-1-AP), STAT1 and phosphorylated STAT1 (CST, #14994; #9167), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: ChIP-sequencing, Co-Immunoprecipitation Assay, Generated, Labeling