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: MSMO1 promotes chemotherapy resistance through modulation of T-MAS metabolism via PERK/elF2α/ATF4/CHOP pathway

doi: 10.1016/j.isci.2026.114790

Figure Lengend Snippet: MSMO1 inhibits cell death mediated by the PERK/eIF2α/ATF4/CHOP pathway (A) Gene set enrichment analysis revealing the pathways significantly changed in MSMO1-KD cells and the control cells. (B) Gene set enrichment analysis revealing the pathways significantly changed in MSMO1-KD cells and the control cells with the pretreatment of 100 nM Tg for 3 h. (C and D) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in MSMO1-KD cells and overexpression cells and the corresponding control cells with or without the pretreatment of 100 nM thapsigargin (Tg) for 3 h. (E) Representative electron microscope images of untreated and Tm-treated (1 μg/mL, 24 h) MSMO1-KD and control cells. Arrowheads indicate the ER. 133 ER widths were measured in each group. x5k scale bars, 5 μm. x25k scale bars, 1 μm. (F) Cell apoptosis analysis of SK-BR-3 MSMO1 overexpressing cells and control cells treated with 100 nM Tg for 48 h. (G) Cell apoptosis analysis of MDA-MB-231 MSMO1-KD cells and control cells treated with 100 nM Tg for 48 h. (H and I) IC50 curves of MSMO1-overexpressing and MSMO1-KD cells, along with the corresponding control cells, treated with Tg, with or without pretreatment with 1 μM GSK2656157 (PERKi) for 3 h. Data are represented as mean ± SEM. (J) Representative images of thioflavin T (ThT) staining assay in MSMO1 overexpressing and control cells with or without the pretreatment of 100 nM Tg for 3 h. Scale bars, 10 μm ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: SK-BR-3 , ATCC , RRID: CVCL_0033.

Techniques: Control, Western Blot, Expressing, Over Expression, Microscopy, Staining

Journal: iScience

Article Title: MSMO1 promotes chemotherapy resistance through modulation of T-MAS metabolism via PERK/elF2α/ATF4/CHOP pathway

doi: 10.1016/j.isci.2026.114790

Figure Lengend Snippet: T-MAS induces ER stress and activates PERK/eIF2α/ATF4/CHOP pathway (A) Diagram of the cholesterol metabolism pathway, using siRNA to inhibit TM7SF2 to reduce the cellular levels of T-MAS. (B and C) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in MSMO1-KD and control cells with or without siRNA interfering with TM7SF2. (D) Diagram of the cholesterol metabolism pathway, using siRNA to inhibit CYP51A1 to reduce the cellular levels of FF-MAS and downstream metabolites. (E) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in wild-type SK-BR-3 cells with or without the pretreatment of 100 nM Tg for 3 h and siRNA interfering with CYP51A1. (F) Western blotting images showing the expression of BIP, PERK, p -eIF2α, ATF4, and CHOP in MSMO1-KD and control MDA-MB-231 cells with or without siRNA interfering with CYP51A1. (G and I) Schematic view of silencing TM7SF2 through siRNA to inhibit the transformation of FF-MAS to T-MAS, followed by the administration of FF-MAS or T-MAS to the cells. (H and J) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, and CHOP with or without siRNA interfering with TM7SF2 and 1 μg/mL FF-MAS(or 1 μg/mL T-MAS) treatment of 48 h.

Article Snippet: SK-BR-3 , ATCC , RRID: CVCL_0033.

Techniques: Western Blot, Expressing, Control, Transformation Assay

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