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: 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: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 overexpression enhances CAR-T cell persistence but impairs in vivo antitumor activity. (A, B) Flow cytometric quantification of GFP + Her2 CAR-T and Her2-IF1 CAR-T cells in mouse peripheral blood mononuclear cells (PBMCs) and spleens. NCG mice (n=4 in each group) were intravenously injected with Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, the peripheral blood and spleen were collected, respectively. The flow cytometry was used to detect the percentage of GFP + CAR-T cells. (C) Tumor growth inhibition by Her2 CAR-T and Her2-IF1 CAR-T cells in SKBR-3 xenografted NCG mice. NCG mice were xenografted with SKBR3 cells (n=5 or 6 in each group). When tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, tumors were excised and weighed. *P < 0.05; **P < 0.01. (D, F) The memory phenotype of Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. (E, G) The exhaustion markers of PD-1, LAG-3 and TIM-3 determination in Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: The human breast cancer cell line SKBR-3 (HTB-30, ATCC) was cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S; Sigma-Aldrich, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Over Expression, In Vivo, Activity Assay, Injection, Flow Cytometry, Inhibition

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: Immunofluorescence and multiplex immunohistochemistry analyses. (A, B) Enhanced CD3 + staining in group of Her2-shIF1 CAR-T in SKBR-3 xenografted tumor tissues. NCG mice were xenografted with SKBR3 cells (n=5 in each group); when tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, or Her2-shIF1 CAR-T cells. Four days later, Mice were euthanized, and tumors were excised for immunofluorescence staining. (C) Multiplex immunohistochemistry staining of 18 formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections, showing ATPIF1 (yellow), CD3 (green), HIF-1α (orange), and DAPI (blue). (D, E) Quantitative analysis of the correlation between CD3 + staining and ATPIF1 + cells (D) , and between HIF-1α + and ATPIF1 + cells (E) from the results of Multiplex immunohistochemistry staining in (C) respectively. **P<0.01, ***P<0.001.

Article Snippet: The human breast cancer cell line SKBR-3 (HTB-30, ATCC) was cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S; Sigma-Aldrich, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Immunofluorescence, Multiplex Assay, Immunohistochemistry, Staining, Injection, Formalin-fixed Paraffin-Embedded

Journal: Cells

Article Title: WBP2 Attenuates Metformin Response in HER2-Positive Breast Cancer Cells by Repressing AMPK Activation and Inducing a Lower AMP:ATP Ratio State Through Enhanced ATP Production

doi: 10.3390/cells15040381

Figure Lengend Snippet: WBP2 expression preferentially inhibits metformin’s anti-cancer efficacy in HER2-positive breast cancer cells. ( A ) A cell viability assay was performed on a panel of eight breast cancer cell lines—SK-BR-3, ZR-75-30, BT-474, MDA-MB-453, MDA-MB-231, MDA-MD-468, BT-549 and BT-20—following 3 days of metformin treatment with varying doses using CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega). ( B ) Immunoblotting analysis showing HER2, WBP2 and β-actin expression in a panel of eight breast cancer cells. Fold change represented refers to WBP2 signals normalized to β-actin and relative to SK-BR-3. ( C ) Heatmap representing WBP2 expression and IC 50 of HER2-positive cells. WBP2 expression fold change is relative to SK-BR-3. IC 50 fold change is relative to SK-BR-3. ( D ) Heatmap representing WBP2 expression and IC 50 of HER2-negative/low cells. WBP2 expression fold change is relative to MDA-MB-468. IC 50 fold change is relative to MDA-MB-231. ( E ) Immunoblotting analysis showing HER2, WBP2 and β-actin expression in a panel of eight breast cancer cells treated with 10 mM metformin for 48 h. Fold change represented is normalized to β-actin and relative to untreated control. ( F – I ) Cell viability assay was performed in breast cancer cell lines with WBP2 overexpressed or knocked down following 5 days of treatment with varying doses of metformin using CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega). ( F ) WBP2 overexpressed in BT-474. ( G ) WBP2 knockdown in SK-BR-3. ( H ) WBP2 knockdown in MDA-MB-231. ( I ) WBP2 knockdown in MDA-MB-468. IC 50 was calculated by non-linear regression using GraphPad Prism 10. The data represent mean ± SEM. ** p < 0.01, *** p < 0.001, NS = not statistically significant; NA = not applicable. EV = Empty Vector control. SCR = Scrambled control. Luc = Luciferase control. Statistical analysis was performed using unpaired Student’s t -test.

Article Snippet: Human breast cancer cell lines SK-BR-3, BT-474, ZR-75-30, MDA-MB-453, MDA-MB-231, MDA-MB-468, BT-549 and BT-20 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life Technologies Corporation, San Diego, CA, USA) supplemented with 10% ( v / v ) Fetal Bovine Serum (FBS) (HyClone-Cytiva, Washington, DC, USA) and 1% ( v / v ) penicillin–streptomycin (Biological Industries, Sartorius, Cromwell, CT, USA.

Techniques: Expressing, Viability Assay, Proliferation Assay, Western Blot, Control, Knockdown, Plasmid Preparation, Luciferase

Journal: Cells

Article Title: WBP2 Attenuates Metformin Response in HER2-Positive Breast Cancer Cells by Repressing AMPK Activation and Inducing a Lower AMP:ATP Ratio State Through Enhanced ATP Production

doi: 10.3390/cells15040381

Figure Lengend Snippet: WBP2 represses metformin-induced AMPK pathway and its associated mTOR inhibition. ( A , B ) Immunoblotting analysis showing AMPK activation and WBP2 expression in cells treated with or without 10 mM metformin for 48 h. ( A ) WBP2 was overexpressed in BT-474 cells using WBP2-expressing plasmid or vector control. ( B ) WBP2 knocked down in SK-BR-3 cells with two different shRNAs or a shSCR control. ( C , D ) Immunoblotting analysis showing AMPK-mTOR signaling components and WBP2 expression in cells treated with or without 10 mM metformin for 48 h. ( C ) WBP2 was overexpressed in BT-474 cells using WBP2-expressing plasmid or vector control. ( D ) WBP2 knocked down in SK-BR-3 cells with two different shRNAs or a shSCR control. GAPDH was used as a loading control. EV = Empty Vector control. SCR = Scrambled control. Fold change represented was normalized to total protein and relative to non-treated EV/shSCR controls.

Article Snippet: Human breast cancer cell lines SK-BR-3, BT-474, ZR-75-30, MDA-MB-453, MDA-MB-231, MDA-MB-468, BT-549 and BT-20 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life Technologies Corporation, San Diego, CA, USA) supplemented with 10% ( v / v ) Fetal Bovine Serum (FBS) (HyClone-Cytiva, Washington, DC, USA) and 1% ( v / v ) penicillin–streptomycin (Biological Industries, Sartorius, Cromwell, CT, USA.

Techniques: Inhibition, Western Blot, Activation Assay, Expressing, Plasmid Preparation, Control

Journal: Cells

Article Title: WBP2 Attenuates Metformin Response in HER2-Positive Breast Cancer Cells by Repressing AMPK Activation and Inducing a Lower AMP:ATP Ratio State Through Enhanced ATP Production

doi: 10.3390/cells15040381

Figure Lengend Snippet: WBP2 enhanced cellular energy status and metabolic function in breast cancer cells. ( A , B ) AMP and ATP were measured from the same samples in breast cancer cells treated with or without 10 mM metformin for 48 h, and ratios were calculated prior to normalization to control conditions. The AMP/ATP ratio fold change was calculated relative to non-treated EV/shSCR controls. Data presented was log 2 of fold change. ( A ) WBP2 was overexpressed in BT-474 cells using WBP2-expressing plasmid or vector control. ( B ) WBP2 was knocked down in SK-BR-3 cells using shWBP2 or shSCR control and re-expressed using WBP2-expressing plasmid or vector control. ( C , D ) Mitochondrial respiration was assessed by measuring oxygen consumption rate (OCR) using the Seahorse XF Mito Stress Test in breast cancer cells treated with or without 10 mM metformin for 48 h. ATP production, basal respiration, maximal respiration, proton leak and spare respiratory capacity were calculated. ( C ) WBP2 was overexpressed in BT-474 cells using WBP2-expressing plasmid or vector control. ( D ) WBP2 was knocked down in SK-BR-3 cells using siWBP2 or siSCR control. ( E , F ) Glycolytic function was assessed by measuring extracellular acidification rate (ECAR) using the Seahorse XF Glycolysis Stress Test in breast cancer cells treated with or without 10 mM metformin for 48 h. Glycolysis, glycolytic capacity and glycolytic reserve were calculated. ( E ) WBP2 was overexpressed in BT-474 cells using WBP2-expressing plasmid or vector control. ( F ) WBP2 was knocked down in SK-BR-3 cells using siWBP2 or siSCR control. EV = Empty Vector control. SCR = Scrambled control. The data represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant. Statistical analysis was performed using GraphPad Prism 10 using one-way ANOVA followed by a post hoc Tukey test.

Article Snippet: Human breast cancer cell lines SK-BR-3, BT-474, ZR-75-30, MDA-MB-453, MDA-MB-231, MDA-MB-468, BT-549 and BT-20 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life Technologies Corporation, San Diego, CA, USA) supplemented with 10% ( v / v ) Fetal Bovine Serum (FBS) (HyClone-Cytiva, Washington, DC, USA) and 1% ( v / v ) penicillin–streptomycin (Biological Industries, Sartorius, Cromwell, CT, USA.

Techniques: Control, Expressing, Plasmid Preparation

Journal: Cells

Article Title: WBP2 Attenuates Metformin Response in HER2-Positive Breast Cancer Cells by Repressing AMPK Activation and Inducing a Lower AMP:ATP Ratio State Through Enhanced ATP Production

doi: 10.3390/cells15040381

Figure Lengend Snippet: RNA sequencing analysis reveals WBP2 regulating a network of genes regulating energy metabolism. RNA sequencing was performed in WBP2 knockdown SK-BR-3 cells with two different shRNA (Sequence #1 and #2) compared to shSCR control. ( A ) Immunoblotting showing WBP2 expression was silenced in WBP2 knockdown SK-BR-3 cells. ( B , C ) Volcano plot of differentially expressed genes (DEG) identified between WBP2 knockdown and control SK-BR-3 cells. Each point represents a gene plotted according to log 2 fold change ( x -axis) and –log 10 ( p -value) ( y -axis). Downregulated gene expression is highlighted in green, upregulated gene expression is highlighted in red, and blue dots represent the gene expression with no change. ( B ) DEGs of knocked down WBP2 with shWBP2#1 compared to shSCR. ( C ) DEGs of knocked down WBP2 with shWBP2#2 compared to shSCR. ( D ) Venn diagram showing DEG between shWBP2#1 and shSCR (red) and DEG between shWBP2#2 and shSCR (blue). The area that intersects represents the common gene sets that were regulated following WBP2 knockdown. ( E ) KEGG pathway enrichment analysis of common downregulated DEGs. The bubble plot displays significantly enriched pathways ranked by Fold Enrichment, with bubble size representing gene count and color indicating –log 10 (FDR). ( F ) Heatmap showing the expression profiles of 14 selected WBP2-downregulated genes involved in energy metabolism and mitochondria function across WBP2 knockdown SK-BR-3 cells. Color intensity expression values represent the log 2 FC. ( G ) Schematic pathway mapping of selected WBP2-downregulated genes summarizing the potential multi-pronged mode of action of WBP2 in regulating energy metabolism. Selected DEGs affected by WBP2 knockdown are highlighted in green. Functions are labelled in Blue. Created with BioRender.com ( H ) Heatmap comparison of selected 14 WBP2-downregulated gene expression profiles shown in ( F ) with that from MDA-MB-231 (TNBC) cells with WBP2 knockdown extracted from another RNA-seq data set. The genes and their respective functions that display differences in the trend of regulation by WBP2 knockdown between the two cell types are highlighted in red. Color intensity expression values represent the log 2 FC.

Article Snippet: Human breast cancer cell lines SK-BR-3, BT-474, ZR-75-30, MDA-MB-453, MDA-MB-231, MDA-MB-468, BT-549 and BT-20 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life Technologies Corporation, San Diego, CA, USA) supplemented with 10% ( v / v ) Fetal Bovine Serum (FBS) (HyClone-Cytiva, Washington, DC, USA) and 1% ( v / v ) penicillin–streptomycin (Biological Industries, Sartorius, Cromwell, CT, USA.

Techniques: RNA Sequencing, Knockdown, shRNA, Sequencing, Control, Western Blot, Expressing, Gene Expression, Comparison