Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure with S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 24 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Bioactive Materials

Article Title: Tailoring nanotopography and antibacterial properties of calcium phosphate bone grafts via fluoride incorporation

doi: 10.1016/j.bioactmat.2025.12.026

Figure Lengend Snippet: (a) Cell proliferation of SaOS-2 cells onto the different CaP discs for 4 h, 7, 14 and 21 days. (b) ALP activity of SaOS-2 cells cultured onto the CaP substrates for 4 h, 7, 14 and 21 days. The same letter indicates no statistically significant differences for the same group at different time points while the same number denotes no statistically significant differences for each time point among all samples. (p < 0.05).

Article Snippet: SaOS-2 cell line (HTB-85), RAW 264.7 cell line (TIB-71), Pseudomonas aeruginosa (ATCC-27853) and Staphylococcus aureus (ATCC-25923) were purchased from American Type Culture Collection (VA, USA).

Techniques: Activity Assay, Cell Culture

Journal: Bioactive Materials

Article Title: Tailoring nanotopography and antibacterial properties of calcium phosphate bone grafts via fluoride incorporation

doi: 10.1016/j.bioactmat.2025.12.026

Figure Lengend Snippet: Merged CLSM images of SaOS-2 cells cultured for 4 h and 7 days on the nanostructured CaP substrates, as well as the Flat and Ti controls. Actin filaments were stained with Alexa Fluor™ 546 phalloidin (orange fluorescence signal) and nuclei with DAPI (blue fluorescence signal).

Article Snippet: SaOS-2 cell line (HTB-85), RAW 264.7 cell line (TIB-71), Pseudomonas aeruginosa (ATCC-27853) and Staphylococcus aureus (ATCC-25923) were purchased from American Type Culture Collection (VA, USA).

Techniques: Cell Culture, Staining, Fluorescence

Journal: Bioactive Materials

Article Title: Tailoring nanotopography and antibacterial properties of calcium phosphate bone grafts via fluoride incorporation

doi: 10.1016/j.bioactmat.2025.12.026

Figure Lengend Snippet: (a) mRNA expression of osteogenic genes ALPL, RUNX2 and SPP1 of SaOS-2 cells cultured directly onto the CaP substrates for 1, 3, 7 and 14 days, determined by real-time PCR (n = 3). (b) Interaction of RAW 246.7 cells with treated discs up to 7 days in cell culture. i) mRNA expression of pro-inflammatory genes TNF, IL1B and IL6 of cells in the direct cell culture, determined by real-time PCR for 1, 3 and 7 days (n = 3). All values are relativized to values of cells at day 1. ii) Protein expression level after 7 days of direct cell culture on the treated discs, measured by inflammation antibody array. Values of protein signal are quantified by image analysis and relativized to control. In Fig. a) and bi), the same letter indicates no statistically significant differences for the same group at different time points while the same number denotes no statistically significant differences for each time point among all samples. (p < 0.05).

Article Snippet: SaOS-2 cell line (HTB-85), RAW 264.7 cell line (TIB-71), Pseudomonas aeruginosa (ATCC-27853) and Staphylococcus aureus (ATCC-25923) were purchased from American Type Culture Collection (VA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Ab Array, Control

Journal: Bioactive Materials

Article Title: Tailoring nanotopography and antibacterial properties of calcium phosphate bone grafts via fluoride incorporation

doi: 10.1016/j.bioactmat.2025.12.026

Figure Lengend Snippet: Co-culture of P. aeruginosa and SaOS-2 cells on the nanostructured CaP discs, as well as Flat and Ti controls. a) Merged CLSM images of: i) a pre-implantation infection model, where the samples were first incubated for 6 h with P. aeruginosa and subsequently SaOS-2 cells were cultured for 24 h; or ii) post-implantation infection model, where SaOS-2 cells were first cultured for 24 h on the substrates, which were subsequently incubated for 6 h with P. aeruginosa . Actin filaments were stained with Alexa Fluor™ 546 phalloidin (orange fluorescence signal) and the nuclei with DAPI (blue fluorescence signal). (b) Orthogonal CLSM images showing simultaneous co-visualization of cells and bacteria stained with Alexa Fluor™ 546 phalloidin (orange fluorescence signal), the nuclei with DAPI (blue fluorescence signal) and SYTO-9 (green fluorescence signal). (c) Dead percentage of P. aeruginosa onto the different CaP substrates and the controls, for both the pre-implantation infection i) and the post-implantation ii) infection models. n = 3; ns and ∗ indicate significance at p > 0.05 and p ≤ 0.05, respectively.

Article Snippet: SaOS-2 cell line (HTB-85), RAW 264.7 cell line (TIB-71), Pseudomonas aeruginosa (ATCC-27853) and Staphylococcus aureus (ATCC-25923) were purchased from American Type Culture Collection (VA, USA).

Techniques: Co-Culture Assay, Infection, Incubation, Cell Culture, Staining, Fluorescence, Bacteria

Journal: Bioactive Materials

Article Title: Ribbon-shaped microgels as bioinks for 3D bioprinting of anisotropic tissue structures

doi: 10.1016/j.bioactmat.2025.12.040

Figure Lengend Snippet: μRB bioinks support multicellular patterning to model breast cancer-bone invasion at tissue interface. (A) Schematic of experimental design: bioprinting of MSCs and osteogenic differentiation to derive bone grid, followed by injection of breast cancer bioink, and monitoring invasion over time using confocal microscopy. (B) Confocal images of MSCs (red) after printing (Scale bar = 1 mm). (C) Confocal images of scaffold sections containing CellTracker-labeled MSCs (red) after 28 days of osteogenic differentiation and GFP + MDA-MB-231 cells extruded into the open pores of the grids (green) (Scale bar = 200 μm). (D) Confocal images of patterned MSC-derived bone (red) with MDA-MB-231 and MCF-7 breast cancer cells (green) after 14 days of co-culture (Scale bar = 1 mm). (E) Quantification of breast cancer cell invasion: percentage that remain in open pores vs. invading into the MSC-bone compartment (n = 5 per group). Values are reported as mean ± S.D. and p-values were determined by two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.005, ∗∗∗∗p ≤ 0.001.

Article Snippet: Breast cancer cell lines MDA-MB-231 (ATCC) and MCF-7 (ATCC) were lentivirus transduced to express GFP and cultured in DMEM media (4.5 g L −1 glucose) supplemented with 10 % (v/v) fetal bovine serum and 1 % (v/v) penicillin-streptomycin.

Techniques: Injection, Confocal Microscopy, Labeling, Derivative Assay, Co-Culture Assay

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Off-target cytotoxicity evaluation of CAR T cells using the 3D GOC system. A) Schematic representation of the differing cytolytic mechanisms of UTD, TV-13, and IL-13 CAR T cells against IL13Rα1 + HT-1080 tumor cells. Created with BioRender.com . B) Flow cytometric analysis confirming IL13Rα1 and mCherry (reporter gene) expression on IL13Rα1 + HT-1080 tumor cells. Antigen expression (IL13Rα1 or mCherry) on viable tumor cells shown in histograms: blue for IL13Rα1 + HT-1080 tumor cells and red for control tumor cells. The values within each histogram indicate the percentage of positive cells, with the mean fluorescence intensity (MFI) shown in parentheses. C) Microfluidic evaluation of off-target toxicities of T cells. (i) Representative tile images of tumor-stroma interface stained for actin cytoskeleton (green), showing differences in migration of IL13R1 + HT-1080 tumor cells (red) within the 3D GOC model across varying densities of UTD, TV-13 CAR, and IL-13 CAR T cells. (ii) Quantification of the migration distance of the IL13Rα1 + HT-1080 tumor cells in response to varying T cell concentrations. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. (iii) Bar graph showing the difference in nuclei per field of view (FOV) across different T cell densities, used as a measure of chain migration by IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (iv) Bar graph representing the percentage of T cells positive for intracellular cytokines in the presence of IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: HT-1080 Culture : Human fibrosarcoma cells (CCL-121, ATCC or HT-1080) were used to generate an off-target cell line (IL13Rα1 + HT-1080) expressing IL13Rα1-T2A-mCherry gene, which was single-sorted for the experiments described here.

Techniques: Gene Expression, Expressing, Control, Fluorescence, Staining, Migration

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Generation of CAR T cell workflow, assessment of CAR transduction, and quantification of on-target antigens on U87. A) Pictographic representation of timeline for CAR T cell culturing and functional assessment. B) Flow cytometric gating strategy of representative donor to quantify CAR transduction applicable to both IL-13 and TV-13 CAR transduced cells. C) Comparative CAR expression distinguished between CD4 and CD8 from a representative donor of Control T Cells (UTD), TV-13, and IL-13 CARs. D) Flow cytometric verification of IL13Rα1 and IL13Rα2 expression on U87 cells.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Transduction, Cell Culture, Functional Assay, Expressing, Control

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: 2D in vitro cytotoxic assessment of CARs polyfunctionality. A) Workflow for intracellular cytokine assay. Created with BioRender.com . B) Flow cytometric gating strategy of the representative donor to identify CAR + T cells from viable singlets. C) Comparative release of IL-2 and TNF-α by CAR + T cells from the representative donor between UTD, TV-13, and IL-13 CAR transduced cells. D ) Comparative release of IFN-γ from the representative between UTD, TV-13, and IL-13 CAR T cells. E ) Graphical display of perforin and granzyme B release ( n = 3 ). ∗ p < 0.05. F) Quantification of the amount of INF-γ released into bulk media across UTD, TV-13, and IL-13 ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. G) Lactate Dehydrogenase (LDH) based quantification rate of tumor lysis across different T cell treatment conditions ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. H) Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis showing the number of intracellular cytokines (TNF-α, IFN-γ, and IL-2) produced per T cell by TV-13 and IL-13 CAR T cells, in response to U87 target stimulation indicating their polyfunctionality. The purple quadrant denotes the percentage of T cells producing all three cytokines, green represents cells producing two cytokines, blue denotes cells producing one, and grey represents cells producing none. Comparable levels of polyfunctionality were observed between the TV-13 and IL-13 groups. Data collected from three biological replicates ( n = 3 ).

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: In Vitro, Cytokine Assay, Lysis, Produced

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Formation of 3D self-assembled microvascular network (μVN) and its influence on U87 cells. A) Establishment of the μVN. (i) Schematic representation detailing the formation of the self-assembled μVN, and (ii) Representative phase contrast tile image of the device showing the progression of μVN formation on day 0 (left) and day 7 (right). B) Characterization of the μVN. (i) 10X tile image of vascular region stained for endothelial marker CD31 (green), junctional protein CD144 (red), and counterstained for nuclei with DAPI (blue) (scale bar: 200 μm), (ii) Phase contrast region of interest (ROI) image highlighting the vascular bundle formed within the vascular region (left), alongside 20X immunofluorescent image showing the expression of CD31(middle), and wrapping of pericytes (α-SMA) around the vascular bundle (right). Scale bars: 100 μm. C) orthogonal sectioning of established μVN confirming the open lumen formation (white arrowhead indicates the open lumen in the orthogonal view). Scale bar: 50 μm. D) Representative immunofluorescent and phase contrast overlap image after injection of 70 kDa fluorescent dextran dye captured at 30s, 1,2, and 4min. Scale bars: 100 μm. E) Line graph image of co-localization of pericytes with endothelial cells based on the scan line (white line) from figure Bii (right). F) Representative immunofluorescent image captured after perfusion of 2 μm fluorescent bead (red) through the CD31 (green) stained vascular bundle. Scale bar: 100 μm. G) Characterization of the μVN in the presence of tumor cells. (i) 10X tile image showing the intact μVN in the vascular (V) region and the migration of the tumor cells (U87-green) from the tumor (T) to the stroma (S) region. Yellow dashed trapezoids and hexagons mark the microposts of the 3D GOC. Scale bar: 200 μm, and (ii) Orthogonal sectioning of the vascular region confirming the maintenance of lumens post U87 injection (white arrowhead indicates the open lumen with white dashed box showing a zoomed-in lumen). Scale bar: 50 μm. Actin acquired with Alexa 647 and CD31 stained with Alexa 555 were pseudo colored in gray and magenta, respectively, for visualization. T, S, V represent the tumor, stroma, and vascular regions of the GOC system.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Marker, Expressing, Injection, Migration

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Evaluation of cytotoxic abilities of T cells against GBM cells within the GOC model. A) Microfluidic 3D invasion assay. (i) Schematic representation depicting the culture of tumor cells with T cells on day 0 (top) and day 3 (bottom), (ii) Representative phase contrast tile image overlapped with GFP (tumor cells) channel captured on day 0 to show the distribution of tumor and T cells across the experimental conditions (Scale bars: 200 μm), and (iii) Representative phase contrast tile image overlapped with GFP channel showing the migration of the U87 cells (green) from the tumor region to the stroma region across three different T cell populations. The densities of U87 are kept consistent across all conditions, and the density of T cells varies from 4 × 10 6 to 15 × 10 6 cells/mL. Images were captured 72 h after the interaction of cells within the GOC model (Scale bars: 200 μm). T-tumor, S-stroma, and V-vascular regions of GOC. B) Assessment of tumor cell migration in the presence of different T cells. (i) Quantification of migration distance from the 3D microfluidic model showing dose-dependent inhibition of U87 migration by the CAR T cells. Data were measured on Day 3 from three biological replicates ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (ii) Comparison of migration distance of the U87 cells in the presence of different concentrations of the T cell population. Analysis performed on samples captured on Day 3 of migration ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. C) xCELLigence-based real-time evaluation of T cell cytolytic capacity. (i) Time-course of the average cell index ( n = 3 donors ) for UTD, TV-13, and IL-13 CAR T cell groups under a 10:1 E: T condition over a 7-day co-culture, measured using the xCELLigence platform, (ii) Bar plot of xCELLigence data comparing averaged cell index values of tumor cells at Day 0 and Day 7 across UTD, TV-13, and IL-13 CAR T cell groups. Data represent mean ± SEM ( n = 3 donors ), ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001, Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) xCELLigence data from a representative donor (Donor 31) showing dose-dependent killing of U87 cells achieved by five doses of TV-13 CAR T cells, and (iv) IL-13 CAR T cells during a 7-day co-culture period.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Invasion Assay, Migration, Inhibition, Comparison, Co-Culture Assay

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Assessment of migratory behavior and proliferative potential of GBM tumor cells in the presence of engineered T cells. A) Evaluation of changes in migratory behavior of tumor cells across UTD, TV-13, and IL-13 T cells based on cytoskeletal organization. (i) Representative tile image of the 3D GOC model stained for actin cytoskeleton (red) showing the tumor-stroma-vascular interface (left), zoomed-in view highlighting the chain migration of the tumor cells from the tumor to the stroma region (middle), 20X region of interest (ROI) showing the disruption in the migratory pattern of the tumor cells and the formation of immune synapse (IS) (right). The white dashed box represents the ROIs alongside an inset image (ROI1) that highlights the formation of multiple IS between the tumor (green) and T cell within the stroma interface. The white arrow shows the IS formation, and the white dashed arrow represents the line scan utilized for intensity profiling to confirm the reorganization of actin cytoskeleton at the tumor-T cell interface . Red- Actin, Green- U87 cells, and DAPI – Blue . Scale bars: 200 μm (left and middle), 50 μm (right). (ii) Quantification of the number of cells migrating in a chain from near and far regions across three different T cell conditions . Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ p < 0.001 , ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) Quantification of the number of cells within a field of view (FOV) from two distinct areas, namely near and far regions, Data are represented as mean ± SD measured from three biological replicated ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. B) Immunofluorescent images of the devices stained for proliferation marker Ki-67. (i) Representative 20X ROI image showing the Ki-67 (red) expression on U87 cells (green) and (ii) Quantification of the number of Ki-67 positive cells across each condition through the proliferative index (Ki-67/Nuclei Ratio), Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗ p < 0.01. One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Migration, Disruption, Marker, Expressing

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