human breast cancer cell lines skbr3  (ATCC)

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Hypoxia promotes enrichment and DNA damage-independent oxidative activation of ataxia telangiectasia mutated. A: Flow cytometry analysis of CD44+/CD24- cell populations in Hs578T and MDA-MB-231 cells after exposure to hypoxia or normoxia; B: Quantitative reverse transcriptase polymerase chain reaction analysis of cancer stem cells-associated genes ( c-Myc , octamer-binding protein 4, Kruppel-like factor 4, sex-determining region Y-box 2, NANOG ) in mammosphere cultures under hypoxia; C-E: Western blot analysis of phosphorylated ataxia telangiectasia mutated, γH2AX, and 53BP1 in Hs578T and MDA-MB-231 cancer stem cells under normoxia, hypoxia, or H 2 O 2 treatment. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01; ns: not significant. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; OCT4: Octamer-binding protein 4; p-ATM: Phosphorylated ataxia telangiectasia mutated.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Activation Assay, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Knockdown, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 promote cancer stem cells enrichment and stemness maintenance in triple-negative breast cancer. A: Effects of serine hydroxymethyltransferase 2 knockdown on mammosphere formation, size, and number in Hs578T and MDA-MB-231 cancer stem cells; B: Effects of methylenetetrahydrofolate dehydrogenase 2 knockdown on mammosphere formation, size, and number; C and D: Western blot analysis of stemness-associated proteins Kruppel-like factor 4 and sex-determining region Y-box 2 after serine hydroxymethyltransferase 2 or methylenetetrahydrofolate dehydrogenase 2 knockdown. Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Knockdown, Western Blot