breast epithelial cell line mcf10a  (ATCC)

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Schematic of the spheroid morphogenesis assay. Single MCF10A/HRAS or MCF10A wild-type cells were cultured in a collagen IV/laminin-rich EHS(Engelbreth-Holm-Swarm) hydrogel to generate basoapically polarized spheroids after 10 days in culture (DiC). (B) Representative immunofluorescence micrographs show differences in basoapical polarization of MCF10A spheroids at 10 DiC depending on HRas activation status. BM (collagen IV, yellow), F-actin cytoskeleton (magenta), nuclei (DAPI, blue) and Golgi protein (GM130, green). (C) HRas activation confirmed by pERK immunofluorescence after 1 hour OHT or EtOH treatment. Representative immunofluorescence intensities of intracellular pERK protein (inverted grey scale) in MCF10A/HRAS spheroids treated with OHT or EtOH for 16 hours. SAC: secondary antibody control. Right, quantification of mean pERK intensity per spheroid (n ≥ 44; 3 independent experiments). Box: interquartile range; whiskers: 5th–95th percentiles; red dots: median. (D) Phase-contrast images show the invasive transition of spheroids (10 DiC) with cell transmigration into the EHS matrix after 65 hours of HRas activation with OHT. EtOH-treated HRas off controls remained non-invasive. Kolmogorov-Smirnov test was performed for the data in C; n.s.: p > 0.05; ****: p ≤ 0.0001. Scale bars: 20 µm (B); 50 μm (C). Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Cell Culture, Immunofluorescence, Activation Assay, Control, Transmigration Assay, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Scheme of BM disruption and cell invasion assay. MCF10A/HRAS spheroids (10 DiC) were isolated from EHS matrix and placed on elastomeric substrates (16 kPa, functionalized with EHS proteins) to count events of local BM rupture and cell transmigration. Mechanical BM stress exertion by breast spheroids at time point of invasion onset was measured by traction force microscopy (TFM): Surface-coupled fluorescent fiducial microbeads were used to track tangential surface deformations from which strain energies were calculated as measure for cell force-generated BM stress. (B) In spheroids, the outer basal cell layer is covered by a BM which itself is in contact to the underlying substrate. Images show the BM integrity of a representative HRas on sample, fixed and stained after adhering (1 hour) to the elastomeric substrate. Collagen IV (yellow), laminin-332 (cyan) and F-actin cytoskeleton (magenta). Zoom in highlights in vivo -like layering of the endogenous BM. (C) Representative sequence of phase-contrast images illustrates the first appearance of protrusive cell bodies (also shown as zoom in), marking onset of BM disruption and cell transmigration. This was counted as a positive event of invasion. (D) Cumulative distribution of BM disruption time, depending on HRas induction on 16 kPa substrates (n ≥ 79 spheroids of ≥ 3 independent experiments). (E) Cumulative distribution of BM disruption time in spheroids treated with blebbistatin for myosin II inhibition and additionally with marimastat for MMP inhibition after HRas induction on stiff 16 kPa substrates (n ≥ 69 spheroids of ≥ 3 independent experiments). (F) Scatter plot shows individual invasion onset time points for the sample conditions analyzed in (D and E) (median and 95% confidence interval (CI)). (G) Calculated strain energies (SE) exerted by individual spheroids at onsets of BM disruption, depending on HRas activation and actomyosin inhibition, (cf. D and E). Representative maps of cell-induced traction stresses per condition from which SE were calculated. Scatter plot: median with 95% CI (n ≥ 48 from 3 independent experiments). Kruskal-Wallis test with Dunn’s multiple comparison test was performed for the data in D and E; n.s.: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Scale bars: 20 µm (B, C and G).

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Disruption, Invasion Assay, Isolation, Transmigration Assay, Microscopy, Generated, Staining, In Vivo, Sequencing, Inhibition, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Representative images of MT1-MMP (inversed grey scale) staining of MCF10A/HRas spheroids incubated with 1 µM OHT or EtOH (HRas off control) for 16 hours, and the secondary antibody control (SAC) to measure unspecific background signals. (B) Quantification of fluorescence intensities of MT1-MMP staining (n = 60 and n = 30 for SAC from three and two individual staining experiments, respectively). Scatter plot includes median and 95% CI. Mann-Whitney-U-test was performed for the data (n.s.: p > 0.05). Scale bars: 20 µm. Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Staining, Incubation, Control, Fluorescence, MANN-WHITNEY, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Schematic of the spheroid morphogenesis assay. Single MCF10A/HRAS or MCF10A wild-type cells were cultured in a collagen IV/laminin-rich EHS(Engelbreth-Holm-Swarm) hydrogel to generate basoapically polarized spheroids after 10 days in culture (DiC). (B) Representative immunofluorescence micrographs show differences in basoapical polarization of MCF10A spheroids at 10 DiC depending on HRas activation status. BM (collagen IV, yellow), F-actin cytoskeleton (magenta), nuclei (DAPI, blue) and Golgi protein (GM130, green). (C) HRas activation confirmed by pERK immunofluorescence after 1 hour OHT or EtOH treatment. Representative immunofluorescence intensities of intracellular pERK protein (inverted grey scale) in MCF10A/HRAS spheroids treated with OHT or EtOH for 16 hours. SAC: secondary antibody control. Right, quantification of mean pERK intensity per spheroid (n ≥ 44; 3 independent experiments). Box: interquartile range; whiskers: 5th–95th percentiles; red dots: median. (D) Phase-contrast images show the invasive transition of spheroids (10 DiC) with cell transmigration into the EHS matrix after 65 hours of HRas activation with OHT. EtOH-treated HRas off controls remained non-invasive. Kolmogorov-Smirnov test was performed for the data in C; n.s.: p > 0.05; ****: p ≤ 0.0001. Scale bars: 20 µm (B); 50 μm (C). Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Cell Culture, Immunofluorescence, Activation Assay, Control, Transmigration Assay, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Scheme of BM disruption and cell invasion assay. MCF10A/HRAS spheroids (10 DiC) were isolated from EHS matrix and placed on elastomeric substrates (16 kPa, functionalized with EHS proteins) to count events of local BM rupture and cell transmigration. Mechanical BM stress exertion by breast spheroids at time point of invasion onset was measured by traction force microscopy (TFM): Surface-coupled fluorescent fiducial microbeads were used to track tangential surface deformations from which strain energies were calculated as measure for cell force-generated BM stress. (B) In spheroids, the outer basal cell layer is covered by a BM which itself is in contact to the underlying substrate. Images show the BM integrity of a representative HRas on sample, fixed and stained after adhering (1 hour) to the elastomeric substrate. Collagen IV (yellow), laminin-332 (cyan) and F-actin cytoskeleton (magenta). Zoom in highlights in vivo -like layering of the endogenous BM. (C) Representative sequence of phase-contrast images illustrates the first appearance of protrusive cell bodies (also shown as zoom in), marking onset of BM disruption and cell transmigration. This was counted as a positive event of invasion. (D) Cumulative distribution of BM disruption time, depending on HRas induction on 16 kPa substrates (n ≥ 79 spheroids of ≥ 3 independent experiments). (E) Cumulative distribution of BM disruption time in spheroids treated with blebbistatin for myosin II inhibition and additionally with marimastat for MMP inhibition after HRas induction on stiff 16 kPa substrates (n ≥ 69 spheroids of ≥ 3 independent experiments). (F) Scatter plot shows individual invasion onset time points for the sample conditions analyzed in (D and E) (median and 95% confidence interval (CI)). (G) Calculated strain energies (SE) exerted by individual spheroids at onsets of BM disruption, depending on HRas activation and actomyosin inhibition, (cf. D and E). Representative maps of cell-induced traction stresses per condition from which SE were calculated. Scatter plot: median with 95% CI (n ≥ 48 from 3 independent experiments). Kruskal-Wallis test with Dunn’s multiple comparison test was performed for the data in D and E; n.s.: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Scale bars: 20 µm (B, C and G).

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Disruption, Invasion Assay, Isolation, Transmigration Assay, Microscopy, Generated, Staining, In Vivo, Sequencing, Inhibition, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Representative images of MT1-MMP (inversed grey scale) staining of MCF10A/HRas spheroids incubated with 1 µM OHT or EtOH (HRas off control) for 16 hours, and the secondary antibody control (SAC) to measure unspecific background signals. (B) Quantification of fluorescence intensities of MT1-MMP staining (n = 60 and n = 30 for SAC from three and two individual staining experiments, respectively). Scatter plot includes median and 95% CI. Mann-Whitney-U-test was performed for the data (n.s.: p > 0.05). Scale bars: 20 µm. Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Staining, Incubation, Control, Fluorescence, MANN-WHITNEY, Imaging

Journal: Frontiers in Pharmacology

Article Title: LITAF suppresses breast cancer and paclitaxel resistance by ubiquitinating and degrading PCMT1 to inhibit COX-2-dependent arachidonic acid metabolism

doi: 10.3389/fphar.2026.1706420

Figure Lengend Snippet: PCMT1 is involved in PTX resistance of BC cells, a process potentially involving COX-2-mediated AA metabolism. (A) RT-qPCR analysis of PCMT1 mRNA levels in tumor tissues and paired adjacent normal tissues from 30 BC patients treated with PTX. (B) Immunoblotting analysis of PCMT1 and COX-2 protein levels in tumor tissues and paired adjacent normal tissues from PTX-treated BC patients (n = 10; 5 PTX-sensitive and 5 PTX-resistant). (C) Correlation between PCMT1 and COX-2 expression was assessed using the Pearson correlation coefficient. (D) RT-qPCR analysis of PCMT1 mRNA levels in MCF10A, BC cells (MDA-MB-231, MCF-7), and PTX-resistant cells (MDA-MB-231/PTX, MCF-7/PTX) (n = 3). (E) Immunoblotting analysis of PCMT1 and COX-2 protein levels in BC cells and PTX-resistant cells (n = 3). (F) IC50 values of BC cells and PTX-resistant cells assessed using the CCK-8 assay (n = 3). Values are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human normal mammary epithelial cells MCF10A (ATCC, USA) were cultured in DMEM/F12 (Procell) supplemented with HS (5%), EGF (20 ng/mL), hydrocortisone (0.5 μg/mL), insulin (0.5 μg/mL), NEAA (1%), and P/S (1%).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Effect of brazilin and derivatives on cell viability of breast cancer and nontumorigenic epithelial cells. Dose–response effects of DMSO and submaximal concentrations of brazilin and derivatives on breast cancer and nontumorigenic cell proliferation. Cell counting assay of proliferating cells treated with increasing concentrations (2.5–40 μM) of brazilin or derivatives is shown for (a-c) MDA-MB-231, (d-f) MCF7, and (g-i) MCF10A cells. Cells cultured in the presence of the DMSO vehicle were used as controls (see Supplementary Figure 1a,b additional DMSO controls). Live cells were quantified by Trypan blue exclusion at 24, 48, 72, and 96 h. The data represents the percentage of cell viability and are plotted as mean ± SE. Data represent three independent biological replicates. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Cell Counting, Cell Culture

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Four-parameter logistic dose–response curve fitting and morphological effects of brazilin and brazilin-(OAc)­3 in breast cell lines. (a–c) Dose–response curves for brazilin in MDA-MB-231 (a), MCF7 (b), and MCF10A (c) cells. (d–f) Dose–response curves for brazilin-(OAc) 3 in MDA-MB-231 (d), MCF7 (e), and MCF10A (f) cells. Curves were generated from cell viability measurements at 48 h and fitted using nonlinear regression (four-parameter logistic model; 4PL for MDA-MB-231 and MCF7, three-parameter logistic model with a fixed slope; 3PL for MCF10A) to estimate the concentration–response relationship and determine IC 50 and IC 10 values, representing the compound concentration required to reduce cell viability by 50% and 10%, respectively. (g) Representative brightfield microscopy images of MDA-MB-231, MCF7, and MCF10A cells after 96 h treatment with 20 μM brazilin or brazilin-(OAc) 3 . Images were captured using a NIKON ECLIPSE Ts2 microscope. Scale bar = 200 μm. Images illustrate representative fields corresponding to the cell counting assays used to generate growth curves. See Supplementary Figure 3 for images at 48 and 72 h. Data are derived from four independent biological replicates ( n = 4) and is plotted as mean ± SE.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Generated, Concentration Assay, Microscopy, Cell Counting, Derivative Assay

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Brazilin and its derivatives modulate apoptosis-related gene and protein expression in breast cell lines. Cells were treated with 20 μM brazilin or its derivatives for 48 h. (a) Steady state mRNA expression analyses was conducted by RT-qPCR to determine the expression of apoptosis-related genes: (a) BAX , (b) BAK , (c) BCL2 , and (d) BCL2L1 . GAPDH was used as control for normalization. Data are presented as mean ± SE from three independent biological replicates and are compared to nontreated control cells. Representative immunoblots and corresponding quantifications of apoptosis-associated proteins are shown for MDA-MB-231 (e–g), MCF7 (h–j), and MCF10A (k–m) cells, including BAX, BCL-2 and cleaved PARP. The BAX/BCL-2 ratio and cleaved PARP levels were quantified and are presented as mean ± SE from three independent biological replicates. Staurosporine (50 nM) was used as a positive control for apoptosis, and GAPDH served as a loading control. Statistical significance was determined relative to untreated cells and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Positive Control

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Antioxidant potential and pro-oxidant effects of brazilin and derivatives in breast cancer and nontumorigenic cells. (a) Radical scavenging activity of brazilin, brazilin-(OMe) 3 , and brazilin-(OAc) 3 (nontreated and 2.5–80 μM) was assessed using DPPH synthetic radicals. The percentage of scavenged radicals is plotted as mean ± SE; derivatives antioxidant potential is compared to brazilin antioxidant potential with a statistical significance of * p < 0.05, ** p < 0.01, *** p < 0.001, data from three independent replicates. (b) Cytoplasmic ROS levels were measured in MDA-MB-231, MCF7, and MCF10A cells after 48 h treatment with 20 μM of each compound by fluorescent detection of DHE oxidation. ( c – h ) Mitochondrial ROS (MitoSox) and mitochondrial membrane potential (TMRE) were evaluated in (c,f) MDA-MB-231, ( d , g ) MCF7, and ( e , h ) MCF10A cells treated for 48 h with 20 μM or 40 μM of brazilin or derivatives. FCCP was used as a positive control for mitochondrial depolarization. The percentage of fluorescence intensity of three independent biological replicates was plotted. Data show means ± SE of three independent biological replicates imaged. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Membrane, Positive Control, Fluorescence

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Validation of differentially expressed genes in breast cancer and nontumorigenic epithelial cells treated with brazilin and derivatives. Differentially expressed genes (DEG) identified by RNA-seq in MDA-MB-231 cells. (a) ATF3 , (b) H2BC21 , (c) SOX4 , (d) MTRNR2L8 , (e) MTRNR2L1 , (f) MTRNR2L2 , and (g) GCLM , were validated by RT-qPCR in MDA-MB-231, MCF7, and MCF10A cells treated with 20 μM brazilin or derivatives for 48 h. Data are expressed as mean ± SE from three independent biological replicates. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR