human tnbc cell lines hs 578 t  (ATCC)

Journal: Cell Death Discovery

Article Title: MSLN-mediated activation of EGFR-ERK1/2 signaling drives liver metastasis in breast cancer

doi: 10.1038/s41420-025-02835-9

Figure Lengend Snippet: A Pearson correlation analysis of the TCGA-TNBC dataset revealed a positive correlation between MSLN and ELF1 expression. B JASPAR database analysis identified the ELF1 recognition motif in the MSLN promoter (upper), and a schematic diagram (lower) illustrated potential ELF1-responsive elements (E1), and introduced an E1 sequence mutation (WT: CAGGAGGAG; Mut: ACAACAACA). C Luciferase reporter assay showed E1-dependent transcription regulation of MSLN by ELF1 in 293 T and HCC1806 Pri cells. D ChIP assays were performed to validate ELF1 binding to the MSLN promoter in 293 T and HCC1806-derived cells. ELF1 regulation of MSLN was analyzed via Luciferase reporter assay ( E ) qRT-PCR ( F ) and WB ( G ) in HCC1806 and 4T1-derived cells. H Pearson correlation analysis of ELF1 and MSLN expression in TNBC patients below median overall survival. (Data are presented as the mean ± SD; ns no statistical difference, * P < 0.05, *** P < 0.001).

Article Snippet: Human TNBC cell lines (HCC1806, MDA-MB-231, Hs578T, BT-549) and mouse breast cancer cell lines (EMT6, 4T1, E0771, PY81119) and 293 T cell line were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Sequencing, Mutagenesis, Luciferase, Reporter Assay, Binding Assay, Derivative Assay, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: MSLN-mediated activation of EGFR-ERK1/2 signaling drives liver metastasis in breast cancer

doi: 10.1038/s41420-025-02835-9

Figure Lengend Snippet: The transcription factor ELF1 in hepatotropic metastatic cancer cells causes an increased MSLN. The elevated MSLN interacts with EGFR, resulting in phosphorylation of EGFR as well as the activation of downstream ERK1/2 signaling pathway, thereby promoting disseminated TNBC cell survival and proliferation, thus lead to liver metastasis.

Article Snippet: Human TNBC cell lines (HCC1806, MDA-MB-231, Hs578T, BT-549) and mouse breast cancer cell lines (EMT6, 4T1, E0771, PY81119) and 293 T cell line were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Phospho-proteomics, Activation Assay

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: IFN-γ promotes PD-L1 nuclear translocation via HDAC2-mediated deacetylation.​​ Western blot analysis of A ​​ total PD-L1 protein levels and B nuclear PD-L1 in MDA-MB-231 cells treated with 0–50 ng/mL human IFN-γ for 48 h. ​​ C Representative Immunofluorescence staining images assessed the subcellular localization of PD-L1 in IFN-γ-stimulated MDA-MB-231 cells (left), colocalization analysis of PD-L1 and DAPI by ImageJ software shown in the right panel.​ D Western blot analysis of cytoplasmic and nuclear fractions from MDA-MB-231 cells treated with 50 ng/mL IFN-γ for 48 h, probed for STAT3 and phosphorylated STAT3. ​​ E ​​ Western blot analysis of cytoplasmic and nuclear PD-L1 in MDA-MB-231 cells treated with 100 µM STAT3 inhibitor S3I-201 and 50 ng/mL IFN-γ for 48 h. ​​ F ​​ Immunoprecipitation (IP) of PD-L1 followed by Western blot for acetylated lysine (Ac-K) in MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. ​​ G ​​ Western blot analysis of nuclear PD-L1 levels in MDA-MB-231 cells treated with 20 µM HDAC2 inhibitor Santacruzamate A or 25 µM Importin α/β inhibitor Ivermectin followed by 50 ng/mL IFN-γ for 48 h. ​​ H ​​ Representative images of lungs and quantification of metastatic nodules from BALB/c mice ( n = 6 per group) fixed in Bouin’s solution 10 days after intravenous injection of 4T1 cells pre-treated with Santacruzamate A for 24 h, followed by IFN-γ treatment for 24 h. 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: The mouse triple-negative breast cancer (TNBC) cell line 4T1 (ATCC ® CRL-2539TM), and human TNBC cell line MDA-MB-231 (ATCC ® HTB-26TM) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS; TransGen) and maintained at 37 °C under 5% CO2.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Staining, Software, Immunoprecipitation, Injection

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: The mouse triple-negative breast cancer (TNBC) cell line 4T1 (ATCC ® CRL-2539TM), and human TNBC cell line MDA-MB-231 (ATCC ® HTB-26TM) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS; TransGen) and maintained at 37 °C under 5% CO2.

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: The mouse triple-negative breast cancer (TNBC) cell line 4T1 (ATCC ® CRL-2539TM), and human TNBC cell line MDA-MB-231 (ATCC ® HTB-26TM) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS; TransGen) and maintained at 37 °C under 5% CO2.

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