Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 binds E2F4 to inhibit its condensate formation. a Py8119 tumor cell lysates were collected and immunoprecipitated with anti-FAM114A1 (left panel), anti-E2F4 (right panel) antibodies, or IgG controls. The interaction between E2F4 and FAM114A1 was determined via western blotting. b , c Py8119 cells with (KD#1 and KD#2) and without (shCoo2) FAM114A1-knockdown were fixed and subjected to immunofluorescent (IF) staining with an anti-E2F4 antibody. DNA was visualized with DAPI ( b ). The percentage of cells with E2F4 condensates was quantified ( c ). Bar, 10 µm. d Py8119 cells were transfected with the E2F4-GFP plasmid. Twenty-four hours after transfection, the cells were fixed for confocal imaging. DNA was visualized with DAPI. Bar, 10 µm. e Py8119 cells were transfected with the indicated E2F4-GFP mutant plasmids. Twenty-four hours after transfection, the cells were fixed for confocal imaging. DNA was visualized with DAPI. Bar, 10 µm. f 293T cells were cotransfected with the FAM114A1-Myc plasmid together with the indicated E2F4-GFP mutants. The cells were collected for co-IP after 24 h. The samples were subjected to western blotting to determine interactions. g Samples from TNBC patients were subjected to IHC staining with an anti-FAM114A1 antibody or IF staining with an anti-E2F4 antibody. DNA was visualized with DAPI. The nuclear areas are highlighted with red dashed circles in inset #1. Representative E2F4 condensates are indicated by red arrows in inset #2. Bar, 10 µm. h FAM114A1 expression was quantified with ImageJ after IHC staining, and the samples were stratified into high- and low-FAM114A1 groups on the basis of median expression. The number of cells with E2F4 condensates was also quantified via IF staining. The data represent the means ± SEMs. P-values were determined via one-way ANOVA ( c ) or the χ 2 test ( h )

Article Snippet: The samples were subsequently centrifuged and subjected to immunoprecipitation (IP) with an anti-E2F4 antibody (10923-1-AP, Proteintech, 5 μg) or an IgG control (provided by the kit).

Techniques: Immunoprecipitation, Western Blot, Knockdown, Staining, Transfection, Plasmid Preparation, Imaging, Mutagenesis, Co-Immunoprecipitation Assay, Immunohistochemistry, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: E2F4 condensates exhibit liquid-like properties and are suppressed by FAM114A1. a Droplet formation by E2F4-GFP was analyzed at the indicated concentrations with 500 mM NaCl at room temperature. E2F4-GFP (5 μM) was examined using droplet formation assays conducted at room temperature ( b , d ) or at 4 °C or 37 °C ( c ) with the indicated concentrations of NaCl ( b ) and with or without 5% Hex at 500 mM NaCl ( c ). For a – d , representative fluorescence and differential interference contrast (DIC) images of the droplets (left of each panel) and quantification of the size of the droplets (right of each panel) are shown. Each dot represents a droplet. Hex, 1,6-hexanediol; Bar, 20 µm. e Live-cell imaging of E2F4-GFP droplets. The arrows indicate representative fused E2F4 condensates. Bar, 10 µm. f E2F4-GFP droplets were subjected to a fluorescence recovery after photobleaching (FRAP) assay. The blue circle indicates the area for photobleaching, and the red circle indicates the area that served as a non-photobleaching control (left panel). The average values for the FRAP data are shown (right panel). Bar, 2 µm. g E2F4-GFP (5 μM) droplet formation was monitored in the absence or presence of the indicated concentrations of GST or GST-FAM114A1 recombinant proteins (left panel). The size of the droplets was quantified (right panel). Bar, 20 µm. The data represent the means and all the data points. The P-value was determined via one-way ANOVA ( g )

Article Snippet: The samples were subsequently centrifuged and subjected to immunoprecipitation (IP) with an anti-E2F4 antibody (10923-1-AP, Proteintech, 5 μg) or an IgG control (provided by the kit).

Techniques: Fluorescence, Live Cell Imaging, Photobleaching Assay, Control, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: E2F4 transcriptionally drives MTDH expression to inhibit antigen presentation. a Py8119-OVA cells with (E2F4-KD#1 and KD#2) or without (shCoo2) stable E2F4 knockdown were cocultured with OT-I splenocytes for 2 h. Tumor cells were collected, and ovalbumin presentation was examined (left panel) and quantified (right panel) via flow cytometry. b scRNA-seq data of cancer cells extracted from TNBC patients. Cells without E2F4 expression were excluded, and the remaining cells were stratified into high- and low-E2F4 expression groups on the basis of the median expression. Antigen presentation signature scores were calculated. c Browser tracks showing the promoter region of MTDH . E2F4 binding motif clusters were identified via MCAST with a motif p-value threshold of 0.005, a maximum gap threshold of 50 bp, and an E-value threshold of 10. d Chromatin immunoprecipitation (ChIP) assays were performed with an anti-E2F4 antibody or an IgG control. The samples were subjected to qPCR with the indicated primers that recognize the MTDH promoter region. e Schematic diagram of the firefly luciferase reporter with wild-type (WT) and mutant (MUT) MTDH promoter regions (−1 to −506 nt) (left panel). WT or MUT reporter plasmids were cotransfected with E2F4 and Renilla luciferase plasmids into Py8119 cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity (right panel). MUT, promoter regions of −131 to −121, −393 to −382, and −412 to −402 were depleted. Py8119-OVA cells with (E2F4-KD#1 and KD#2) or without (shCoo2) stable E2F4 knockdown were collected for RT‒qPCR or western blotting to detect E2F4 or MTDH at the mRNA ( f ) or protein level ( g ), respectively. The data represent the means ± SEMs. P-values were assessed using one-way ANOVA ( a , d , f ) or two-tailed Student’s t -test ( b , e )

Article Snippet: The samples were subsequently centrifuged and subjected to immunoprecipitation (IP) with an anti-E2F4 antibody (10923-1-AP, Proteintech, 5 μg) or an IgG control (provided by the kit).

Techniques: Expressing, Immunopeptidomics, Knockdown, Flow Cytometry, Binding Assay, Chromatin Immunoprecipitation, Control, Luciferase, Mutagenesis, Activity Assay, Western Blot, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 enhances E2F4-mediated MTDH expression. a Py8119 cells were subjected to IF staining with an anti-E2F4 antibody. DNA was visualized with DAPI. The cells and nuclei are highlighted with white and red dashed circles, respectively. Bar, 10 µm. b The fluorescence intensity of nuclear E2F4 in Py8119 (left panel) and TNBC patient samples (right panel) was quantified. n = 50 cells in each group. c , d Py8119-OVA-Luc cells with (KD#1 and KD#2) or without (shCoo2) stable FAM114A1-knockdown were used to extract cytosolic or nuclear proteins. The expression of E2F4 and FAM114A1 in the cytosol and nucleus was examined via western blotting ( c ). The cells were subjected to RNA and protein extraction. The mRNA and protein levels of FAM114A1 and MTDH were examined via RT‒qPCR and western blotting, respectively ( d ). e RNA sequencing data of TNBC patients from the TCGA dataset were extracted. The Spearman rank correlation between MTDH and FAM114A1 was plotted. n = 124 TNBC patients. f , g TNBC patient samples were employed for IHC staining with anti-FAM114A1 and anti-MTDH antibodies. Representative images are shown ( f ). The expression levels of FAM114A1 and MTDH were quantified with ImageJ and analyzed with Spearman’s rank correlation ( g ). n = 109 TNBC patients. Bar, 500 µm. The data represent the means ± SEMs. P-values were determined by two-tailed Student’s t -test ( b ) or one-way ANOVA ( d )

Article Snippet: The samples were subsequently centrifuged and subjected to immunoprecipitation (IP) with an anti-E2F4 antibody (10923-1-AP, Proteintech, 5 μg) or an IgG control (provided by the kit).

Techniques: Expressing, Staining, Fluorescence, Knockdown, Western Blot, Protein Extraction, RNA Sequencing, Immunohistochemistry, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1-mediated immunosuppression is independent of its ability to regulate the cell cycle. a‒c Py8119-OVA-Luc tumor cells with FAM114A1-knockdown and the corresponding controls were used for the tumor sphere assay. Five days after culture, the spheres were collected for cell cycle and apoptotic analysis ( a ), and the proportion of cells in each phase and the proportion of apoptotic cells were quantified ( b , c ). n = 3 replicates per group. shCoo2: Py8119-OVA-Luc PLKO-shCoo2; KD#1: Py8119-OVA-Luc PLKO-shFAM114A1#1; KD#2: Py8119-OVA-Luc PLKO-shFAM114A1#2. d , e Py8119 tumor cells with and without FAM114A1-knockdown were orthotopically injected into nude mice. Tumor size and weight were evaluated at the endpoint ( d ). n = 7 and 8 mice for the control (shCoo2) and FAM114A1-knockdown (KD) groups, respectively. Tumors from the indicated groups were collected for IHC staining with anti-Ki67 and cleaved caspase 3 (CC-3) antibodies. The percentage of positive cells was determined ( d ). n = 5 tumors per group. f Py8119 cells were synchronized with a double thymidine block. The cells released at the indicated time points were subjected to flow cytometry analysis to examine the cell cycle distribution and antigen presentation status. asy. : asynchronized. g Cells at the indicated phases were subjected to immunofluorescence staining with an anti-E2F4 antibody. The percentage of cells with E2F4 condensates at the indicated phases was quantified (left panel). The antigen presentation status of cells at the indicated phases was quantified (right panel). h OT-I female mice were pretreated with 125 μg/mouse anti-CD8 antibody or the corresponding isotype control every two days for one week. Py8119-OVA-Luc tumor cells with/without FAM114A1-knockdown were orthotopically injected into the mice, and the mice were continually treated with 125 μg/mouse of anti-CD8 antibody or the corresponding isotype control twice per week until the end of the experiment. The tumor size was measured 6 weeks after tumor cell injection. Primary tumors were dissected and weighed at week 7. n = 6 mice for the FAM114A1 control (shCoo2)- and isotype (IgG)-treated groups; n = 8 mice for the FAM114A1-knockdown (KD)- and isotype (IgG)- or anti-CD8 antibody-treated groups. The data represent the means ± SEMs. P-values were determined via one-way ANOVA ( b , c , g , h ) or two-tailed Student’s t -test ( d , e )

Article Snippet: The samples were subsequently centrifuged and subjected to immunoprecipitation (IP) with an anti-E2F4 antibody (10923-1-AP, Proteintech, 5 μg) or an IgG control (provided by the kit).

Techniques: Knockdown, Injection, Control, Immunohistochemistry, Blocking Assay, Flow Cytometry, Immunopeptidomics, Immunofluorescence, Staining, Two Tailed Test