mouse breast cancer cell lines 4t1  (ATCC)

Journal: Animal Models and Experimental Medicine

Article Title: Characterizing a murine breast cancer mouse model reveals chromosomal abnormalities in structure and number of single‐cell clones and the presence of rare cancer stem cell‐like phenotypes

doi: 10.1002/ame2.70156

Figure Lengend Snippet: A 4T1 BALB/c mouse model of breast cancer. The left flanks of mice were injected with 4T1 cells subcutaneously. (A) A representative picture of an injected mouse is shown with a white arrow indicating the site of primary tumor growth. (B) The tumor volume was evaluated twice per week. (C) A representative excised tumor. (D) H&E staining of primary tumor tissue at the end point of tumor development with pleomorphic vesicular nuclei (green arrows) and hyperchrome prominent nucleoli (yellow arrows) indicated (40× magnification, and the scale bar represents 20 μm).

Article Snippet: The 4T1 mouse breast cancer cell line was purchased from the American Type Culture Collection (ATCC R CRL‐2539) (Manassas, Virginia, USA).

Techniques: Injection, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Characterizing a murine breast cancer mouse model reveals chromosomal abnormalities in structure and number of single‐cell clones and the presence of rare cancer stem cell‐like phenotypes

doi: 10.1002/ame2.70156

Figure Lengend Snippet: Chromosome counts of metaphase cells. (A) Chromosome number of murine 4T1 breast cancer cells prepared ex vivo (23 cells) and in vitro (100 cells). Boxes represent data range from 1st quartile to 3rd quartile. Whiskers represent the range of chromosomes from minimum to the maximum. (B) Descriptive statistics and Mann–Whitney two‐tailed test results. (C) Distribution of chromosome numbers in ex vivo and in vitro cells.

Article Snippet: The 4T1 mouse breast cancer cell line was purchased from the American Type Culture Collection (ATCC R CRL‐2539) (Manassas, Virginia, USA).

Techniques: Ex Vivo, In Vitro, MANN-WHITNEY, Two Tailed Test

Journal: Animal Models and Experimental Medicine

Article Title: Characterizing a murine breast cancer mouse model reveals chromosomal abnormalities in structure and number of single‐cell clones and the presence of rare cancer stem cell‐like phenotypes

doi: 10.1002/ame2.70156

Figure Lengend Snippet: Analysis of breast cancer stem cell‐like phenotypes (CD44 + /CD24 − ). (A) Flow cytometric analysis of CD24/CD44 expression in ex vivo and in vitro cells. (B) Percentage of four subpopulations in breast cancer ex vivo and in vitro 4T1 cells. Standard deviation is indicated for each respective cell suspension, n = 3.

Article Snippet: The 4T1 mouse breast cancer cell line was purchased from the American Type Culture Collection (ATCC R CRL‐2539) (Manassas, Virginia, USA).

Techniques: Expressing, Ex Vivo, In Vitro, Standard Deviation, Suspension

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: ZBTB21 deficiency activates pyroptosis through the NLRP3/GSDMD axis to enhance immunotherapy response. (A) Schematic description of the selection of ZBTB21 from multiple databases: Prognosis: TCGA (hazard ratio > 1.5, p < 0.05); refractory to PD‐1 blockade: GEO: GSE78220 ( p < 0.05; |log2fold change (FC)| > 1.2); T Cell State Score (Quiescence): TCGA (Pearson's R > 0.1; p < 0.05); Pyroptosis related transcription factors: ChIP‐Atlas, KnockTF, and JASPAR databases. (B) Negative correlation between ZBTB21 and GSDMD ‐mRNA expression levels in tumor samples from TCGA‐BRCA dataset. (C) Kaplan‐Meier overall survival curves of TNBC patients with ZBTB21 low ( n = 621) versus ZBTB21 high ( n = 304). ZBTB21 ‐mRNA expression of the auto‐selected best cutoff. (D) Kaplan–Meier overall survival curves of Melanoma patients receiving anti‐PD‐1 treatment with ZBTB21 low ( n = 123) versus ZBTB21 high ( n = 155). ZBTB21 ‐mRNA expression of the auto‐selected best cutoff. (E) Analysis of ZBTB21 gene expression across diverse cell types within various tumor using the pan‐cancer single‐cell sequencing data. (F) The images show representative micrographs of pyroptosis induced by Nigericin (NIG), where pyroptotic cells are stained red with propidium iodide (PI). Yellow arrows indicate key pyroptotic features. Scale bar: 20 µm. (G,H) Relative levels of GSDMD and NLRP3 ‐mRNA expression measured by qPCR ( n = 3). (I) Immunoblotting results showing the expression of the indicated proteins in ZBTB21 ‐KO and Control 4T1 cells treated with or without NIG. (J,K) IL‐1β and IL‐18 concentration in supernatant of ZBTB21 ‐KO and Control 4T1 cells treated with or without NIG ( n = 3). (L) Transcriptomic analysis of ZBTB21 ‐KO versus Control 4T1 cells. GSEA plots corresponding to each Hallmark pathway, displaying the normalized enrichment score (NES), nominal p value (Nominal P), and FDR (FDR q). Significantly enriched pathways were defined as those with |NES| > 1, nominal p value < 0.05, and FDR q value < 0.25. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data were analyzed by one‐way ANOVA, Log‐rank (Mantel‐Cox) test or Spearman test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Selection, Expressing, Gene Expression, Single Cell, Sequencing, Staining, Western Blot, Control, Concentration Assay

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: ZBTB21 loss activates GSDMD pyroptosis to enhance CD8 + T cell immunity and amplify checkpoint blockade. (A,B) Tumor growth and overall survival curves for 4T1 Control and ZBTB21 ‐KO tumor‐bearing mice ( n = 7). (C,D) Tumor growth and overall survival curves for tumor‐bearing mice implanted with ZBTB21 ‐KO cells rescued by ZBTB21‐ cDNA or empty vector (EV) ( n = 6). (E,F) Tumor growth curves for B16F10 ( n = 6) or HEPA1‐6 ( n = 7) Control and ZBTB21 ‐KO tumor‐bearing mice. (G) Relative levels of GSDMD and NLRP3 ‐mRNA expression measured by qPCR in tumor cells (CD45 − CD31 − CD90.2 − cells) isolated from 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice ( n = 4). (H) IL‐1β and IL‐18 concentration in serum from 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice ( n = 6). (I) Immunoblotting results showing the expression of the indicated proteins of tumor cells isolated from 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice ( n = 2). (J) Representative staining profiles (left) of CD4/CD8 and percentages (right) of CD8 + T cells among CD45 + cells in 4T1 Control or ZBTB21 ‐KO tumors ( n = 5). (K) Heatmap based on percentages of the indicated immune cell populations among CD45 + cells in 4T1 Control or ZBTB21 ‐KO tumors ( n = 4). (L) Association between ZBTB21 expression and CD8 + T cells infiltration across human cancers (TCGA dataset). (M) Representative staining profiles (left) and percentages (right) of IFN‐γ and Perforin expression in tumor‐infiltrating CD8 + T cells from 4T1 Control and ZBTB21 ‐KO tumors ( n = 5). (N) Representative staining profiles (left) and percentages (right) of PD‐1 and Tim3 expression in tumor‐infiltrating CD8 + T cells from 4T1 Control and ZBTB21 ‐KO tumors ( n = 5). (O) Immunoblotting results for T cell activation signaling pathway‐related protein phosphorylation in tumor‐infiltrating CD8 + T cells isolated from 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice. (P) Tumor growth curves for 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice treated with αCD8 or IgG antibodies ( n = 5). (Q,R) Tumor growth and overall survival curves for 4T1 Control or ZBTB21 ‐KO tumor‐bearing mice treated with αPD1 or IgG antibodies ( n = 5). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data were analyzed by two‐way ANOVA, Log‐rank (Mantel‐Cox) test, unpaired two‐tailed Student's t ‐test or Spearman test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Control, Plasmid Preparation, Expressing, Isolation, Concentration Assay, Western Blot, Staining, Activation Assay, Phospho-proteomics, Two Tailed Test

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: ZBTB21 loss enhances tumor immunogenicity and response to immunotherapy through MHC‐I upregulation. (A) Association between ZBTB21 expression and HLA/B/C across human cancers (TCGA dataset). (B) Transcriptomic analysis of ZBTB21 ‐KO versus Control 4T1 cells. GSEA plots corresponding to each Hallmark pathway, displaying the normalized enrichment score (NES), nominal p value (Nominal P), and FDR (FDR q). A Hallmark pathway was considered significantly enriched if it met the following criteria: |NES| > 1, nominal p value < 0.05, and FDR q value < 0.25. (C) Representative staining profiles (left) and H‐2Kb SIINFEKL MFI (right) in Control+OVA and ZBTB21 ‐KO+OVA cells ( n = 5). (D) Cytotoxicity analysis of splenic OT‐I T cells cocultured with OVA‐expressing B16F10 tumor cells versus ZBTB21 ‐KO B16F10 tumor cells ( n = 5). (E) Representative staining profiles (left) and IFN‐γ detection results (right) of T cells following co‐culture with ZBTB21 ‐KO MDA‐MB‐231 cells or parental MDA‐MB‐231 cells ( n = 4). (F,G) Tumor growth curves ( n = 5) and overall survival curves ( n = 7) of tumor‐bearing mice inoculated with Control, ZBTB21 ‐ B2m ‐KO, or ZBTB21 ‐KO tumor cells. (H,I) Tumor growth curves for Control, ZBTB21 ‐ B2m ‐KO, or ZBTB21 ‐KO tumor tumor‐bearing mice treated with PD1 or CTLA‐4 antibodies ( n = 5). (J) Tumor growth ( n = 5) and overall survival ( n = 6) curves for Control, ZBTB21 ‐ B2m ‐KO, or ZBTB21 ‐KO tumor‐bearing mice treated with OVA‐encoding mRNA‐LNP vaccine or empty‐vector mRNA‐LNP as control. (K) Representative staining profiles (left) of CD8 and percentages (right) of CD8 + T cells among CD45 + cells in Control, ZBTB21 ‐ B2m ‐KO, or ZBTB21 ‐KO tumors ( n = 5). (L) Representative staining profiles (left) and IFN‐γ MFI (right) in Control, ZBTB21 ‐ B2m ‐KO, or ZBTB21 ‐KO tumors ( n = 5). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance. Data were analyzed by two‐way ANOVA, Log‐rank (Mantel‐Cox) test or unpaired two‐tailed Student's t ‐test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Immunopeptidomics, Expressing, Control, Staining, Co-Culture Assay, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: ZBTB21 deficiency upregulates GSDMD via facilitating STAT1‐driven transcription by enhancing chromatin accessibility. (A) Profile around the TSS of H3K27ac‐modified genes. Read counts were extracted for all CUT&Tag‐seq experiments within a region spanning ± 3 kb around the TSS. (B) IGV analysis of H3K27ac peaks at the GSDMD locus with the indicated scale in 4T1 Control and ZBTB21 ‐KO cells. (C) GO and KEGG analyses of genes containing modified regions of H3K27ac. The biological process terms of interest are displayed. The size of a circle indicates the number of enriched genes, and the color reflects the adjusted p value (|log2FC| >1 and FDR < 0.05). (D) Distribution of ATAC‐seq signals in the region upstream and downstream of the GSDMD gene transcription start site (TSS). (E) ChIP‐qPCR analysis of H3K27ac antibody‐pulled down chromatins (n = 3). (F) ChIP‐qPCR analysis of p‐STAT1 antibody‐pulled down chromatins ( n = 3). (G,H) Relative levels of GSDMD ‐mRNA expression measured by qPCR ( n = 3). (I) Immunoblotting for STAT1 in 4T1 Control and ZBTB21 ‐KO cells with or without IFN‐γ. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance. Data were analyzed by two‐way ANOVA or unpaired two‐tailed Student's t ‐test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Modification, Control, ChIP-qPCR, Expressing, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: ZBTB21 represses MHC‐I antigen presentation through epigenetic silencing of IRF1. (A) IGV analysis of H3K27ac peaks at the IRF1 locus with the indicated scale in 4T1 Control and ZBTB21 ‐KO cells. (B) Distribution of ATAC‐seq signals in the region upstream and downstream of the IRF1 gene transcription start site (TSS). (C) ChIP‐qPCR analysis of H3K27ac antibody‐pulled down chromatins ( n = 3). (D) Relative levels of IRF1 ‐mRNA expression measured by qPCR ( n = 3). (E) Immunoblotting for IRF1 in 4T1 Control and ZBTB21 ‐KO cells. (F) ChIP‐qPCR analysis of IRF1 antibody‐pulled down chromatins. (ISRE, 5'‐GAAANNGAAA‐3'). (G,H) Effect of ZBTB21 overexpression or Knockout on IRF1‐induced activation of the MHC‐I promoter. (I) Relative levels of MHC‐I ‐mRNA expression measured by qPCR ( n = 3). (J) Representative staining profiles (left) and H‐2Kd/Dd MFI (right) in ZBTB21 ‐KO cells treated with IRF1‐siRNA or negative control (NC) ( n = 5). (K,L) Co‐immunoprecipitation analysis of protein complexes associated with ZBTB21. (M) Representative staining profiles (left) and H‐2Kb SIINFEKL MFI (right) in Control+OVA and ZBTB21 ‐KO+OVA cells ( n = 5). (N) Relative levels of MHC‐I ‐mRNA expression measured by qPCR ( n = 3). (O) Immunoblotting for MHC‐I in 4T1 Control and IRF1 ‐KO cells. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data were analyzed by unpaired two‐tailed Student's t ‐test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Immunopeptidomics, Control, ChIP-qPCR, Expressing, Western Blot, Over Expression, Knock-Out, Activation Assay, Staining, Negative Control, Immunoprecipitation, Two Tailed Test

Journal: Advanced Science

Article Title: ZBTB21 Is a Dual Suppressor of Pyroptosis and MHC‐I Antigen Presentation That Promotes Tumor Immune Evasion

doi: 10.1002/advs.202519836

Figure Lengend Snippet: Dobutamine targets ZBTB21 to attenuate transcriptional repression and enhance antitumor immunity. (A) The AlphaFold‐modeled structure of ZBTB21 was refined. (B) Schematic workflow of the docking‐based virtual screening for small‐molecule inhibitors of ZBTB21. (C) Relative levels of ZBTB21 ‐mRNA expression measured by qPCR in 4T1 cells treated with or without Cefoperazone (Cefo), Iopamidol (Iopa), or Dobutamine (Dobu) ( n = 3). (D) The binding affinity between Dobu and ZBTB21 was determined by surface plasmon resonance. (E) ZBTB21 mRNA decay rate measured by quantitative PCR in cells cultured with Dobu and actinomycin D. (F) Tumor growth curves of 4T1 tumor‐bearing mice treated with or without Dobu ( n = 6). The results for Figure , Figure originated from the same set of experiments. (G) Tumor growth curves of 4T1 tumor‐bearing mice treated with or without Dobu, αCD8, or IgG antibodies ( n = 6). The results for Figure , Figure originated from the same set of experiments. (H) Representative staining profiles (left) of CD4/CD8 and percentages (right) of CD8 + T cells among CD45 + cells in 4T1 tumor‐bearing mice treated with or without Dobu ( n = 5). (I) Representative staining profiles (left) and percentages (right) of IFN‐γ and Perforin expression in tumor‐infiltrating CD8 + T cells in 4T1 tumor‐bearing mice treated with or without Dobu ( n = 5). (J) Relative levels of ZBTB21 ‐mRNA expression measured by qPCR in sorted tumor cells from 4T1 tumor‐bearing mice treated with or without Cefo, Iopa, or Dobu ( n = 3). (K) Representative staining profiles (left) and MFI (right) of H‐2Kd/Dd expression in tumor‐infiltrating CD8 + T cells in 4T1 tumor‐bearing mice treated with or without Dobu ( n = 5). (L–N) Molecular docking and dynamics simulations assessing the binding mode of Dobu with ZBTB21. (O–Q) The stability of the Dobu‐ZBTB21 binding was assessed through root‐mean‐square deviation (N), radius of gyration (O), and root‐mean‐square fluctuation (P). (R,S) Tumor growth and overall survival curves for 4T1 tumor‐bearing mice treated with or without Dobu, αPD1, or IgG antibodies ( n = 6). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance. Data were analyzed by one‐way ANOVA, two‐way ANOVA, Log‐rank (Mantel‐Cox) test or unpaired two‐tailed Student's t ‐test.

Article Snippet: Mouse triple‐negative breast cancer cell line 4T1 (Cat# CRL‐2539), mouse melanoma cell line B16F10 (Cat# CRL‐6475), mouse hepatocellular carcinoma cell line Hepa1‐6 (Cat# CRL‐1830), human triple‐negative breast cancer cell line MDA‐MB‐231 (Cat# CRM‐HTB‐26), human malignant melanoma cell line A375 (Cat# CRL‐1619), human non‐small cell lung cancer cell line H1299 (Cat# CRL‐5803), and human embryonic kidney 293T (Cat# CRL‐3216) cells were obtained from the American Type Culture Collection.

Techniques: Expressing, Binding Assay, SPR Assay, Real-time Polymerase Chain Reaction, Cell Culture, Staining, Two Tailed Test