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: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: SL-II CD16 + neutrophils retain tumor cell killing capacity but are deficient in immunosuppressive capacity (A) Cartoon of antibody dependent cellular cytotoxicity (ADCC) and myeloid derived suppressor cell (MDSC) activity exerted by mature activated neutrophils (Figure adapted from Aarts et al. ). (B) In vitro ADCC of LAN-1 cells unopsonized and opsonized (+Dinutuximab) ( n = 4) and SKBR3 cells unopsonized and opsonized (+Trastuzumab) by PMNs (blue) ( n = 6) and SL-II CD16 + neutrophils (pink) ( n = 3) in a 1:50 T:E ratio. (C) Representative CFSE plots for CD8 + T cell proliferation of an in vitro MDSC activity assay. T cells were stimulated with anti-CD3/CD28 antibodies to induce proliferation and co-cultured with either unstimulated or TNFα-stimulated PMNs or SL-II CD16 + neutrophils. After 4 days, T cell proliferation was assessed by CFSE dilution ( n = 12 for T cells alone and n = 16 for co-culturing with PMNs or SL-II CD16 + neutrophils). (D) Boxplots of CD8 + T cell proliferation and damaged T cell formation in the in vitro MDSC assay. Unstimulated or TNFα stimulated PMNs (blue) ( n = 12) or SL-II CD16 + neutrophils (pink) ( n = 16) were co-cultured with T cells. After 4 days, FSC/SSC gating was used to assess ‘damaged’ T cell formation next to T cell proliferation. (E) Representative images taken with imaging flow cytometry of trogocytosis carried out by PMNs and SL-II CD16 + neutrophils. The scale bar was set at 10 μm ( n = 3). PMNs and SL-II CD16 + neutrophils were stained with calcein red-orange (orange), and T cells were stained with DiD (red) before co-culturing for 4 h. Data in (B) and (D) is represented as median and interquartile range. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. n values represent the number of individual donor samples.

Article Snippet: Human Breast Cancer Cell Line SKBR3 , ATCC , .

Techniques: Derivative Assay, Activity Assay, In Vitro, Cell Culture, Imaging, Flow Cytometry, Staining, MANN-WHITNEY, Labeling

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: In A) Sk-BR-3 human HER2-positive breast-carcinoma cells were stimulated with epidermal growth factor (EGF; 50 ng/ml) for 60 min, or left either serum-starved (Starvation control) or in complete-media control conditions; whole-cell lysate from K562 cells was loaded as a positive control in the right-most lane. Immunoblots were probed sequentially for STAT3 (≈70 kDa; Abcam, ab171359, 1:2 000), STAT5A (≈80 kDa; Abcam, ab213219, 1:1 000), ERK2 (≈42 kDa; Thermo Fisher, PA5-29636, 1:1 000), MEK1 (≈45 kDa; Abcam, ab239802, 1:1 000), AKT2 (≈55 kDa; Thermo Fisher, PA5-85518, 1:1 000), phospho-STAT3-Tyr705 (pSTAT3, ≈90–115 kDa; R&D, AF4607, 1:2 000) and pan-AKT (≈60 kDa; CST, #4691, 1:1 000). Membranes were finally reprobed for GAPDH (≈37 kDa; CST, #14C10, 1:1 000) and vinculin (≈120 kDa; CST, #E1E9V, 1:1 000) as loading controls. Molecular-weight markers are indicated on the left. For all panels, proteins were resolved on Bis-Tris SDS–PAGE gels (4–12 % or 10 % as specified in Methods) using MES or MOPS running buffer, transferred to PVDF, and detected by near-infrared fluorescence on a LI-COR Odyssey® imager. In B) estimated relative band intensities of key signaling proteins in Sk-BR-3 and K562 cells are shown. Fluorescence-based Western blot images (Supp (A)) were used to visually estimate band intensities of STAT3, phospho-STAT3 (Tyr705), STAT5A, ERK2, MEK1, AKT2, and pan-AKT in Sk-BR-3 cells under three conditions: serum starvation, complete media, and 60 min EGF treatment, and in K562 cells as a positive control. Band intensities were approximated by visual comparison of signal brightness and normalized to the corresponding housekeeping protein (GAPDH or vinculin) within each lane. Normalized values were plotted as grouped bar graphs to highlight relative abundance patterns across conditions. This semi-quantitative representation supports qualitative comparison and was not derived from raw densitometry. validates the antibody specificity and establishes the baseline expression of PLA target proteins in Sk-BR-3 and K562 cells. Panel A displays the Western blot images of Sk-BR-3 cells subjected to three conditions: serum starvation, complete media (control), and EGF stimulation for 60 minutes. K562 cell lysate is included as a positive control. Panel B presents the quantified band intensities, normalised to the housekeeping proteins GAPDH or vinculin and expressed as percentages. In Sk-BR-3 cells, total STAT3, ERK2, MEK1, AKT2, and pan-AKT were consistently detected across all conditions. STAT3 levels remained stable, while phosphorylation at Tyr705 (pSTAT3) dropped to undetectable levels under serum starvation, rose slightly under media control, and reached higher levels after 60 min of EGF treatment. This trend suggests growth-factor-responsive STAT3 activation despite unchanged total STAT3 levels. MEK1 and AKT2 expression decreased mildly in media control compared to both EGF and starvation conditions, while ERK2 and pan-AKT remained constant. STAT5A was not detected in any Sk-BR3 condition. In contrast, the K562 lysate showed robust STAT5A expression, confirming its hematopoietic lineage specificity. STAT3 was present at lower levels relative to Sk-BR-3 media control, while ERK2 and pan-AKT were comparable between both cell types. MEK1 and AKT2 appeared at slightly lower levels in K562 cells. GAPDH and vinculin levels were consistent across all samples, confirming equal loading and transfer quality. Together, these results confirm the specificity of each antibody, the absence of non-specific binding, and the suitability of both Sk-BR-3 and K562 cells as models for downstream proximity ligation assays aimed at monitoring dynamic signalling interactions.

Article Snippet: The SkBr3 human breast cancer cell line (ATCC, HTB-30), derived from pleural effusion, was maintained according to standard procedures.

Techniques: Control, Positive Control, Western Blot, Molecular Weight, SDS Page, Fluorescence, Comparison, Derivative Assay, Expressing, Phospho-proteomics, Activation Assay, Binding Assay, Ligation