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

Journal: Scientific Reports

Article Title: Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression

doi: 10.1038/s41598-023-40347-7

Figure Lengend Snippet: Sortilin (SORT) interacts with progranulin (PRGN) and LPL in PCa cells and PRGN is secreted by aggressive PC3 cells. ( a ) Representative confocal images showing co-labelling of cells with anti-PRGN (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( b ) Quantification of colocalisation between PRGN and SORT. ( c ) Amount of PRGN in conditioned media (CM) and corresponding cell lysates with quantification of band densities and ratio of PRGN in CM to PRGN in cell lysates. ( d ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and PRGN was detected by Western blotting. Input; cell lysate. ( e ) Representative confocal images showing co-labelling of cells with anti-LPL (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( f ) Quantification of colocalisation between LPL and SORT. ( g ) Amount of LPL in CM and corresponding cell lysates with quantification of band densities in cell lysates. In ( c ) and ( g ) PNT2 bands from the same membrane were shifted towards LNCaP and PC3 bands. Western blotting signal was normalised using total protein staining. ( h ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and LPL was detected by Western blotting. Input; cell lysate. Data are presented as mean ± SD, in ( b , f ) n = 10 representative ROIs, in ( c , g ) n = 3 (independent experiments), one-way ANOVA.

Article Snippet: Human prostate cell PNT2 (ECACC 95012613), LNCaP (clone FCG, ECACC 89110211) and PC3 (ECACC 90112714) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) from CellBank Australia (Children's Medical Research Institute, Westmead, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Membrane, Staining