brightvision anti rabbit ap  (Vector Laboratories)

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: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Validation of mutant selectivity of KRAS G12V siRNA in vitro (A) UV melting curves and sequences of 23mer duplexes between the fully modified KRAS guide RNA and the targeted G12V mutant, WT, and G12D mutant RNA. (B) Western blot analysis in A431 cells stably expressing KRAS WT or G12V transiently transfected with siRNAs. Cells were analyzed at 48 hours and 72 hours. Blots were done separately, and densitometry quantification below is based on vinculin control for each individual blot. (C) RT-qPCR analysis in A431 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 48 hours. Data shown as mean +/- SEM, experiments performed in duplicate. (D) Luciferase dose-response curve in A431-KRAS-WT or A431-KRAS-G12V cells stably expressing a luciferase reporter. Cells were analyzed at 72 hours. Data shown as mean +/- SEM, experiments performed in triplicate. (E) Volcano plots from RNA-sequencing in SKCO1 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 24 hours. This figure includes data published in Stanland et al and has received permission to be shown in this figure.

Article Snippet: SKCO1 , ATCC , Cat #HTB-39; RRID: CVCL_0626.

Techniques: Biomarker Discovery, Mutagenesis, In Vitro, Modification, Western Blot, Stable Transfection, Expressing, Transfection, Control, Quantitative RT-PCR, Luciferase, RNA Sequencing