Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: Characterization of YAP/β-catenin expression and proliferation rate in breast cancer cells when subject to different stiffness (A) Random field of view images were taken under brightfield with a magnification of 20× of MCF-7 and MDA-MB-231 cells. Scale bars = 100 μm (B) Proliferation rates of MCF-7 and MDA-MB-231 cells on the substrate with different stiffness were measured using alamarBlue Cell viability reagent over the course of 8 days. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ∗ p < 0.05, ∗∗∗∗ p < 0.0001. (C) Western blot results of YAP and β-catenin in MCF-7 and MDA-MB-231 cells that were subject to 2 kPa and 32 kPa substrate. Protein expression of β-catenin, YAP, and pYAP was analyzed with GAPDH used for normalization.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: YAP controls actin cytoskeletal organization and polymerization in a stiffness-dependent manner in metastatic breast cancer cells (A) MCF-7 and MDA-MB-231 cells were transfected with non-targeting control siRNAs (control) or siRNA against YAP (siYAP) for 48 h. Control group was treated with LatA at a concentration of 0.4 μM for 1 h (LatA). After the fixation, permeabilization, and blocking, the cells were stained for F-actin and G-actin. Images were taken using Cytation5 under 20× magnification. Scale bars = 50 μm (B) The quantification of the F/G actin ratio in MCF-7 cells (left) and MDA-MB-231 cells (right) from the images in A. Image analysis was performed using BioTek Gen5 Software, and bar graphs were created using GraphPad Prism. Data represent mean ± SD. n = individual cells pooled from 5 randomly acquired images per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Transfection, Control, Concentration Assay, Blocking Assay, Staining, Software

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: Enhanced translocation of YAP and β-catenin to the nucleus occurs in response to a more rigid substrate in metastatic breast cancer cells (A) The intracellular distribution of β-catenin and YAP in MCF-7 and MDA-MB-231 cells, subjected to the substrates of 2 kPa and 32 kPa, was examined through immunofluorescent staining. Scale bars = 100 μm (B) YAP (top) and β-catenin (bottom) nuclear translocation was measured using BioTek Gen5 Software, and results were generated using GraphPad Prism. Data represent mean ± SD. n = individual cells pooled from 15 images (5 fields × 3 wells) per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗∗∗∗ p < 0.0001.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Translocation Assay, Staining, Software, Generated

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: β-catenin nuclear translocation following YAP knockdown is regulated by cell density in metastatic breast cancer cells (A) YAP and β-catenin in MDA-MB-231 cells at low, medium, and high confluency after YAP knockdown. Low confluency (30% confluency), medium confluency (50% confluency), and high confluency (80% confluency). MDA-MB-231 cells that were subject to 2 kPa and 32 kPa substrates were transfected with non-targeting siRNA (control) or siRNA against YAP (siYAP) before the immunofluorescent staining for YAP and β-catenin. Scale bars = 100 μm (B) The percentages of the cell population that show β-catenin nuclear translocation in A were measured in Agilent BioTek Gen 5 and plotted in GraphPad Prism. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01. (C) The analysis of β-catenin and its active isoform expression in MCF-7 and MDA-MB-231 cells on 2 kPa and 32 kPa substrates following YAP knockdown. Total protein amount was used for normalization, and bar graphs were generated using GraphPad Prism. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ns: not significant.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Translocation Assay, Knockdown, Transfection, Control, Staining, Expressing, Generated

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: β-catenin nuclear translocation upon YAP depletion supports cell proliferation and migration in metastatic breast cancer cells in a stiffness-dependent manner (A) Proliferation analysis of MCF-7 and MDA-MB-231 cells following YAP knockdown. Both MCF-7 cells and MDA-MB-231 cells were transfected with non-targeting siRNA (control), siRNA against YAP (siYAP), or siRNA against β-catenin (siβ-catenin). Then alamarBlue assay was performed for a period of 7 or 10 days at each time point for both MCF-7 cells and MDA-MB-231 cells. Then the data were generated using GraphPad Prism. Data represent mean ± SD. n = 3 wells per condition. (B) Cell migration analysis of MDA-MB-231 cells that were cultured on 2 kPa or 32 kPa substrates before the transfection with non-targeting siRNA (control), or siRNA against YAP (siYAP), or siRNA against YAP and siRNA against β-catenin (siYAP and siβ-catenin). Then the gap closure assay was performed, and the gap areas were imaged using Cytation5 under 4× magnification at 0 h and 20 h. Scale bars = 1000 μm (C) Quantitative analysis of the cell migration assay in B was conducted using ImageJ, and the results were generated using GraphPad Prism. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using one-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗ p < 0.05.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Translocation Assay, Migration, Knockdown, Transfection, Control, Alamar Blue Assay, Generated, Cell Culture, Cell Migration Assay

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: Matrix stiffness modulates total and phosphorylated β-catenin levels in breast cancer cells (A) Representative Western blots of total β-catenin and phosphorylated β-catenin at S33/S37/T41 and S675 in MCF-7 and MDA-MB-231 cells cultured on soft (2 kPa) and stiff (32 kPa) substrates. (B) Quantification of protein levels normalized to loading controls (mean ± SD). Top row: Total β-catenin expression showed no significant differences across stiffness in MCF-7 cells but was significantly increased in MDA-MB-231 cells cultured on stiff (32 kPa) compared to soft (2 kPa) substrates (∗ p < 0.05). Middle row: Phosphorylation of β-catenin at S33/S37/T41 (associated with degradation) was significantly higher in MDA-MB-231 cells than in MCF-7 cells at both stiffness levels (∗∗ p < 0.01), indicating enhanced β-catenin degradation in the metastatic subtype. However, substrate stiffness had no significant effect on p-β-catenin (S33/S37/T41) within either cell line. Bottom row: Phosphorylation at S675 (associated with nuclear translocation) was significantly higher in MCF-7 cells than in MDA-MB-231 cells at both stiffness levels ∗∗∗ p < 0.001). Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using two-tailed t-tests. ns: not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Western Blot, Cell Culture, Expressing, Phospho-proteomics, Translocation Assay, Two Tailed Test

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: YAP knockdown selectively downregulates AXIN2 expression on soft substrates in metastatic breast cancer cells (A) Capillary-based immunoblot and quantification confirming efficient YAP knockdown in MDA-MB-231 cells cultured on soft (2 kPa) and stiff (32 kPa) substrates for 48 h. YAP protein levels were significantly reduced in siYAP-treated cells compared to control (∗∗∗∗ p < 0.0001). Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using two-way ANOVA followed by the Tukey post hoc test. (B) Immunoblot and quantification of CCND1 and AXIN2 protein expression in MDA-MB-231 and MCF-7 cells following YAP knockdown. In MDA-MB-231 cells, YAP depletion did not significantly affect CCND1 expression at either stiffness, while AXIN2 levels were significantly decreased under 2 kPa (∗ p < 0.05) but not 32 kPa. In MCF-7 cells, CCND1 expression was significantly elevated on stiff substrate compared to soft (∗ p < 0.05), independent of YAP knockdown. AXIN2 expression in MCF-7 cells was not significantly affected by YAP knockdown at either stiffness. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using two-way ANOVA followed by the Tukey post hoc test. ns: not significant.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Knockdown, Expressing, Western Blot, Cell Culture, Control

Journal: iScience

Article Title: A stiffness-gated YAP-β-catenin axis orchestrates AXIN2 expression in metastatic breast cancer

doi: 10.1016/j.isci.2025.114405

Figure Lengend Snippet: β-catenin knockdown reduces CTGF mRNA but not protein expression, and does not affect CCND1 expression (A) qPCR analysis showing efficient CTNNB1 knockdown and the CCND1 and CTGF transcript levels in MDA-MB-231 and MCF-7 cells. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using two-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (B) Western blots and quantification confirm reduced β-catenin protein following siRNA treatment, but no significant change in CCND1 or CTGF protein expression. These results suggest that β-catenin alone is insufficient to regulate YAP target genes in the absence of YAP. Data represent mean ± SD. n = 3 wells per condition. Statistical significance was assessed using two-way ANOVA followed by the Tukey post hoc test. ns: not significant, ∗∗∗∗ p < 0.0001.

Article Snippet: MCF-7 human breast cancer cell line , ATCC , HTB-22; RRID: CVCL_0031.

Techniques: Knockdown, Expressing, Western Blot

Journal: Human Genetics and Genomics Advances

Article Title: Understanding exceptional response: The role of MINDY1 SNP in CDK4/6 inhibitor therapy for ER + , HER2 − advanced breast cancer

doi: 10.1016/j.xhgg.2025.100560

Figure Lengend Snippet: CRISPR-spCas9-mediated engineering of breast cancer cell models (A) Schematic illustration of the CRISPR-spCas9-mediated homology-directed repair strategy used to introduce the rs771205 SNP into the MINDY1 locus of MCF-7 cells. (B) Sanger sequencing of RT-PCR amplicon from parental MCF-7 cells showing the SNP at position c.130 of MINDY1 transcript. (C) Sanger sequencing of RT-PCR amplicon from CRISPR-edited MCF-7 cells confirming successful SNP edit at position c.130 of the MINDY1 transcript. (D) Western blot shows MINDY1 protein levels in ExR (c.130G) and RefG (c.130A) cell lines. (E) Real-time qPCR indicates MINDY1 mRNA expression levels in ExR and RefG cell lines. Error bars indicate the standard deviation (SD). Statistical significance was assessed using a two-tailed Student’s t test.

Article Snippet: ER + human breast cancer cell line MCF-7 (American Type Culture Collection [ATCC], catalog no. HTB-22) and its derivative, MCF-7 MINDY1 ( motif interacting with Ub-containing novel DUB family-1 ) c.130A, were maintained at 37°C, 100% humidity, and 5% CO 2 .

Techniques: CRISPR, Introduce, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Expressing, Standard Deviation, Two Tailed Test