4t1 triple negative breast cancer cells  (ATCC)

Journal: Cancer Research

Article Title: ACSL5 Mediates Adaptation to the Palmitic Acid–Enriched Pulmonary Microenvironment to Enhance Metastatic Breast Cancer Cell Survival and Lung Metastasis

doi: 10.1158/0008-5472.CAN-25-0866

Figure Lengend Snippet: ACSL5 is highly expressed in lung metastatic breast cancer cells and fostered lung metastasis. A, Eight metabolism-related genes are commonly upregulated in lung metastatic breast cancer cells. B, Western blotting was used to evaluate ACSL5 protein levels in primary tumor cells (PRI) and organ-specific derivatives (left), as well as in the lung metastatic derivatives (right) of MDA-MB-231. LM1, the first generation of lung metastasis; LM2, the second generation of lung metastasis; LM3, the third generation of lung metastasis, breast cancer cells with lung-preferential metastasis. C and D, Representative images ( C ) and quantification ( D ) of IHC of ACSL5 protein levels in primary tumors (PRI) and lung metastases of patients with breast cancer ( n = 7). E, ACSL5 mRNA levels in lung, liver, brain, and bone metastases of patients with breast cancer ( GSE14020 ). F, Kaplan–Meier survival curves for overall survival of patients with breast cancer with low and high ACSL5 mRNA levels ( GSE14020 ). G, Representative images of BLI. BALB/c nude mice were injected with MDA-MB-231 and HCC1806 cells. BALB/c mice were injected with 4T1 cells. All data represent the mean ± SD. Student t test ( D ), Kruskal–Wallis test ( E ) and log-rank test ( F ) were utilized. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Triple-negative breast cancer cell lines 4T1, MDA-MB-231, and HCC1806 and mouse alveolar epithelial cell line MLE-12 were purchased from the ATCC.

Techniques: Western Blot, Injection

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Schematic representing the time course of conditioned media (CM) collection for a 4T1 TNBC cell scratch assay. (B and C) Representative images of 4T1 TNBC cells in scratch assays at the 0 and 15 h time points following incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 6) is shown in (C). (D) Schematic representing the time course of the scratch assay with 4T1 TNBC cells co-cultured with either irradiated or unirradiated 3T3 fibroblasts. (E and F) Representative images of 4T1 TNBC cells (red) in scratch assays co-cultured with irradiated or unirradiated 3T3 fibroblasts (unlabeled) at the 0 and 25 h time points. The corresponding quantification (n = 3–4) is shown in (F). (G) Schematic representing the time course of CM collection for the 3D 4T1 TNBC cell tumorsphere assay. (H and I) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 30 tumorspheres) is shown in (I). (J and K) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated shScr or shAtg5 fibroblast CM. The corresponding quantification ( n = 18 tumorspheres) is shown in (K). (L and M) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated fibroblast CM with or without an IL-6 neutralizing antibody. The corresponding quantification ( n = 25–26 tumorspheres) is shown in (M). (N) Schematic representing the time course of shScr and shAtg5 fibroblast irradiation and co-culture with 4T1 TNBC cells in the tumorsphere assay. (O and P) Representative images of 4T1 TNBC tumorspheres after 10 days of co-culture with irradiated or unirradiated shScr or shAtg5 fibroblasts. The corresponding quantification ( n = 16 tumorspheres) is shown in (P). Statistical analysis was performed as follows: for (C) and (F), an unpaired two-tailed t test with * p < 0.05 and ** p < 0.01; for (I), a Mann-Whitney test for non-normally distributed distributions with ** p < 0.01; and for (K), (M), and (P), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bars are 200 μm in (B) and (E) and 500 μm in (H), (J), (L), and (M). Error bars show standard deviation.

Article Snippet: 4T1 murine triple negative breast cancer cells , ATCC , Cat#CRL-2539; RRID:CVCL_0125.

Techniques: Wound Healing Assay, Incubation, Irradiation, Control, Cell Culture, Co-Culture Assay, Two Tailed Test, MANN-WHITNEY, Standard Deviation

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: We demonstrate that RT induces increased autophagic flux, mitochondrial fusion, and lipid droplet accumulation up to 7 days after treatment. Increased mitochondrial fusion and FAO contribute to the maintenance of high levels of respiration in irradiated fibroblasts. Mitochondrial fusion is disrupted when autophagy is blocked, resulting in decreased respiration. Over time, mitochondria generate increased ROS, signifying increased mitochondrial and cellular stress. This coincides with an increase in inflammatory cytokines, such as IL-6, leading to an altered secretory profile. Irradiated fibroblasts induce an aggressive phenotype in TNBC cells, and pro-metastatic tumor cell behavior is abrogated through either blocking autophagy in irradiated fibroblasts or neutralizing IL-6.

Article Snippet: 4T1 murine triple negative breast cancer cells , ATCC , Cat#CRL-2539; RRID:CVCL_0125.

Techniques: Irradiation, Blocking 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 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

Journal: The Journal of Biological Chemistry

Article Title: TIM-3 inhibition enhances breast tumor progression and metastasis: A paradoxical immune checkpoint response

doi: 10.1016/j.jbc.2025.111096

Figure Lengend Snippet: Treatment with anti-TIM3 monoclonal antibody increase tumor growth and weight. A , treatment schema. Six weeks old female Balb/c mice were inoculated with 4T1 (1.2 × 10 6 ) at the fourth abdominal mammary duct of the left flank (n = 3/group). Anti-TIM3 antibody (250 μg/injection) was given i.p. on day 18,21, 24 and day 28. B and C , in vivo tumor development at day 40. B , 4T1 induced control breast tumor ( C ) Breast tumor after anti-TIM3 administration. D , representative photograph of the visceral organs of Tumor mice and anti-TIM3 treated mice collected on day 40 after tumor inoculation. E , tumor length and width were measured on day 8, 16, 21, 24, 32 and until day 40 and tumor volume were determined. Bar plot represents mean ± SD. ∗∗ p < 0.01 (Unpaired t test with Holm-Šídák method). F , tumor weight was measured on day 40, the day of resection of each group of mice. Data represent mean ± SD. ∗∗∗ p < 0.001 (Unpaired two tailed t test).

Article Snippet: Murine triple-negative breast cancer (TNBC) cell line 4T1 was purchased from ATCC (ATCC, cat. #CRL-2539, RRID: CVCL_0125) and cultured in RPMI-1640 (Himedia, cat. #AL162S) supplemented with 10% fetal bovine serum (Himedia, cat. #RM112) and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO 2 and humidification.

Techniques: Injection, In Vivo, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: TIM-3 inhibition enhances breast tumor progression and metastasis: A paradoxical immune checkpoint response

doi: 10.1016/j.jbc.2025.111096

Figure Lengend Snippet: Histopathological and immunohistochemical evaluation of TIM-3 into 4T1 allograft breast tumors and detection of liver metastases following anti-TIM-3 monoclonal antibody treatment. A and B , TIM-3 immunohistochemical detection mostly observed into tumor infiltrating immune cells (indicated by red arrow ) in primary breast tumor tissue into both control tumor mice and anti-TIM3-treated mice, respectively, at × 400 magnification with a scale bar 20 μm. C , TIM-3 expression scoring into tumor (Control) and anti-TIM-3 treated group was determined semi-quantitatively by counting the percentage of TIM-3-positive cells and staining intensity of five × 400 magnification fields selected randomly and represented in the bar graph as mean ± SD, from three mice per group, ∗∗∗ p < 0.001 (Unpaired two-tailed t-test). D-G , primary breast tumor tissue section stained with H&E, ( D and E ) breast tumor tissue histology of control tumor mice and anti-TIM3 treated mice (at × 100 magnification respectively, with scale bar 100 μm), and ( F and G ) breast tumor tissue histology of control tumor mice and anti-TIM3 treated mice respectively (at × 400 magnification respectively, with scale bar 100 μm) resected after day 40. H – L , histological analysis of liver metastasis of tumor-bearing mice and treated group showed ( H ) tumor foci (indicated with a black circle ) of control tumor mice, and ( I ) anti-TIM3 mAb-treated tumor mice showed several tumor foci (indicated with several black circles ). J , the number of tumor metastatic foci into liver tissue section was determined by counting fifty high power field under × 400 magnification of each mouse of an experimental group (n = 3). Data represent mean ± SD. ∗ p < 0.05 (Unpaired two-tailed t -test). Infiltration of inflammatory cells into liver was observed and demarcated by ( K ) blue arrow indicated the moderate (not too severe) infiltration into control tumor-bearing mice, while ( L ) the green arrow indicated the severe inflammatory cell infiltration into the liver of anti- TIM-3 treated group. All data represent mean ± SD; n = 3.

Article Snippet: Murine triple-negative breast cancer (TNBC) cell line 4T1 was purchased from ATCC (ATCC, cat. #CRL-2539, RRID: CVCL_0125) and cultured in RPMI-1640 (Himedia, cat. #AL162S) supplemented with 10% fetal bovine serum (Himedia, cat. #RM112) and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO 2 and humidification.

Techniques: Immunohistochemical staining, Control, Expressing, Staining, Two Tailed Test