Journal: Science Advances

Article Title: The risk variant rs11836367 contributes to breast cancer onset and metastasis by attenuating Wnt signaling via regulating NTN4 expression

doi: 10.1126/sciadv.abn3509

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) analysis of genes preranked by Pearson correlation to NTN4 expression for four breast cancer–related signatures, which were BENPORATH_PROLIFERATIION [red curve: normalized enrichment score (NES) = −3.87, false discovery rate (FDR) q value < 0.001], POOLA_INVASIVE_BREAST_CANCER_UP (blue curve: NES = −2.34, FDR q value < 0.001), VANTVEER_BREAST_CANCER_POOR_PROGNOSIS (yellow curve: NES = −1.81, FDR q value = 0.001), and ZHANG_BREAST_CANCER_PROGENITORS_UP (purple curve: NES = −2.01, FDR q value = 0.001) in RNA sequencing (RNA-seq) datasets from TCGA. ( B ) Schematic of dCas9-SAM–mediated doxycycline (Dox)–inducible endogenous overexpression of NTN4 . TetR, Tet repressor; IRES, internal ribosomal entry site; 5’LTR, 5’ long terminal repeat; PuroR, puromycin resistent gene. ( C ) Validation of inducible overexpression of the NTN4 protein by Western blot analysis in SKBR3-dCas9-VP64-MPH and MDA-MB-231-dCas9-VP64-MPH cells following the addition of Dox (50 ng/ml). AC-sg1, AC-sg2, and AC-sg3 were designed to target the NTN4 promoter, and notargeting AC-sgNC was a negative control. ( D ) Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) after 4-day culture with or without Dox treatment. OD 450 , optical density at 450 nm. ( E ) Cellular migration was analyzed using the Transwell assay with or without Dox treatment. ( F ) Cellular invasion was investigated using the three-dimensional spheroid invasion assay in Matrigel following a 6-day culture with or without Dox treatment. ( G ) Cancer stem cell features were analyzed using sphere formation assays after a 7-day culture with or without Dox treatment. ( H ) Validation of CRISPRi knockdown of NTN4 protein by Western blot analysis in SUM159-dCas9-KRAB cells. KB-sg1, KB-sg2 and KB-sg3 were designed to target the NTN4 promoter, and nontargeting KB-sgNC was used as a negative control. ( I to L ) Cellular proliferation (I), migration (J), invasion (K), and cancer stem cell features (L) were analyzed in NTN4 knockdown SUM159-dCas9-KRAB cell lines as described above. Data are represented as means ± SEM of three to six independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Related representative images for (E) to (G) and (J) to (L) were shown in fig. S4.

Article Snippet: The SUM159 cell line was purchased from Asterand (Detroit, MI, USA).

Techniques: Expressing, RNA Sequencing, Over Expression, Biomarker Discovery, Western Blot, Negative Control, Cell Counting, CCK-8 Assay, Migration, Transwell Assay, Invasion Assay, Knockdown

Journal: Breast Cancer Research : BCR

Article Title: Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells

doi: 10.1186/s13058-015-0662-4

Figure Lengend Snippet: The effect of Ixabepilone or Cetuximab on cell viability of triple-negative breast cancer cell lines with WST-1. WST-1 proliferation assay of MDA-MB-231 cells and SUM159 cells treated with Cetuximab (MDA-MB-231 cells ( a ) SUM159 cells ( b )), or Ixabepilone (MDA-MB-231 cells ( c ), SUM159 cells ( d )). The data for each cell line are mean ± standard deviation obtained from three independent experiments

Article Snippet: MDA-MB-231 or SUM159 cells (2 × 10 6 cells/100 ml) were injected orthotopically in female immunocompromised severe combined immunodeficiency (SCID)/Beige mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Proliferation Assay, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells

doi: 10.1186/s13058-015-0662-4

Figure Lengend Snippet: FACS analysis for double staining of CD44 + /CD24 -/low and Aldefluor ( ALDF ) in MDA-MB-231, or Aldefluor in SUM159. Representative results of FACS analysis identified by CD44 + /CD24 -/low in MDA-MB-231 ( a ), Aldefluor + in MDA-MB-231 ( b ), and Aldefluor + in SUM159 ( c ) treated with different doses of Cetuximab (10 μg/ml, 30 μg/ml, or 50 μg/ml) for 3 days. Proportion (%) of CD44 + /CD24 -/low or Aldefluor + cells were determined and represented by the average of FACS analysis performed in triplicate

Article Snippet: MDA-MB-231 or SUM159 cells (2 × 10 6 cells/100 ml) were injected orthotopically in female immunocompromised severe combined immunodeficiency (SCID)/Beige mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Double Staining

Journal: Breast Cancer Research : BCR

Article Title: Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells

doi: 10.1186/s13058-015-0662-4

Figure Lengend Snippet: Mammosphere-forming efficiency of MDA-MB-231 and SUM159 cells treated with Cetuximab +/- Ixabepilone. Mammosphere formation (%) of MDA-MB-231 ( a ) and SUM159 ( b ) cells was compared between control (no treatment) and Cetuximab treatment at concentrations of 10 μg/ml, 30 μg/ml, 50 μg/ml or between Ixabepilone (5 nM) and Ixabepilone (5 nM) + Cetuximab treatment at 10 μg/ml, 30 μg/ml, 50 μg/ml. The Mann–Whitney U test was used to determine the p values. Data represent the mean of three independent experiments. N.S. not significant

Article Snippet: MDA-MB-231 or SUM159 cells (2 × 10 6 cells/100 ml) were injected orthotopically in female immunocompromised severe combined immunodeficiency (SCID)/Beige mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Control, MANN-WHITNEY

Journal: Breast Cancer Research : BCR

Article Title: Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells

doi: 10.1186/s13058-015-0662-4

Figure Lengend Snippet: MDA-MB-231 and SUM159 tumor models of xenograft mice treated with Cetuximab, Ixabepilone, and combination. Means of the percentage in tumor volume of control (intravenous (i.v.), PBS), Cetuximab (i.v., 4 mg/kg/weekly), Ixabepilone (intraperitoneal (i.p.), 10 mg/kg/weekly), or combination (Cetuximab, i.v + Ixabepilone, i.p (equivalent dose/weekly))-treated MDA-MB231 ( a ) or SUM159 ( b ) tumors in SCID/Beige mice were plotted as a function of time. Two-way analysis of variance with Bonferroni post-hoc test was used to determine significant differences among groups and p <0.05 was considered to be statistically significant

Article Snippet: MDA-MB-231 or SUM159 cells (2 × 10 6 cells/100 ml) were injected orthotopically in female immunocompromised severe combined immunodeficiency (SCID)/Beige mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Control

Journal: Breast Cancer Research : BCR

Article Title: Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells

doi: 10.1186/s13058-015-0662-4

Figure Lengend Snippet: FACS analysis and mammosphere-forming efficiency in MDA-MB-231 or SUM159 xenografts. Representative results of CD44 + /CD24 -/low population (%) in MDA-MB-231 xenografts ( a ), Aldefluor + population (%) in SUM159 xenografts ( b ), mammosphere formation (%) in MDA-MB-231 xenografts ( c ), or mammosphere formation (%) in SUM159 xenografts ( d ). CD44 + /CD24 -/low population (%) and mammosphere formation (%) of MDA-MB-231 xenografts were reduced by Cetuximab alone or combination treatment. Aldefluor + population (%) in SUM159 xenografts decreased by combination treatment when compared to control. CD44+/CD24- cells (%) in SUM159 xenografts were extremely low in proportion and are not represented in the figure. N.S. not significant

Article Snippet: MDA-MB-231 or SUM159 cells (2 × 10 6 cells/100 ml) were injected orthotopically in female immunocompromised severe combined immunodeficiency (SCID)/Beige mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Control

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a , b Detection of the EGFR/HER2 dimer using FSEC. GFP- and mCherry-tags are attached to the C-termini of EGFR and HER2, respectively. HER2_DT is the tail-deleted form with a substituted MBP-tag. EGFR_JM and HER2_JM constructs only contain the ectodomain, transmembrane domain, part of the juxtamembrane domain, and the coiled-coil (CC) peptide. The basic and acidic CC peptides are attached to EGFR_JM and HER2_JM, respectively. The red pentagram in b indicates the peak position of EGFR_JM/HER2_JM dimer. c Cryo-EM map of the EGF-bound EGFR/HER2 ectodomain complex shown in two views. EGFR, EGF, and HER2 are colored in green, marine, and magenta, respectively. d Overall structure of the EGF-bound EGFR/HER2 heterodimer in ribbon presentation. The color code is the same as that in c . The four domains of EGFR and HER2 ectodomains are indicated with Roman numerals. The DAs of Domain II are also labeled.

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Construct, Cryo-EM Sample Prep, Labeling

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a Superposition of the individual EGFR subunit structures in different dimers. EGFR in our EGFR/HER2 dimer (green) is superimposed with the bent EGFR protomer structures in EGF (cyan; PDB code: 1IVO)-, TGFα (orange; PDB code: 1MOX)-, and EREG (magenta; PDB code: 5WB7)-bound EGFR dimers. The RMSDs between our EGFR and the other three structures are 1.82 Å, 2.20 Å, and 1.72 Å, respectively. b Superposition of the individual HER2 subunit structures. HER2 in our EGFR/HER2 dimer (magenta) is overlaid with its monomer structure (rat) (cyan; PDB code: 1N8Y), as well as its structure in complex with Pertuzumab Fab (orange; PDB code: 1S78) or HER3 (green; PDB code: 7MN5). The RMSDs between our HER2 and the other three structures are 1.41 Å, 2.15 Å, and 1.34 Å, respectively. c Superposition of our EGFR/HER2 structure (blue) with the currently reported three asymmetric HER dimers (golden), namely, NRG1β-bound HER2/HER3 (left; PDB code: 7MN5; RMSD 1.95 Å), EREG-bound EGFR (middle; PDB code: 5WB7; RMSD 1.73 Å), and Spitz-bound Drosophila EGFR (dEGFR) (right; PDB code: 3LTG; RMSD 2.68 Å). d Cryo-EM map of the EREG-bound EGFR/HER2 ectodomain complex. EGFR, EREG, and HER2 are colored green, light blue, and magenta, respectively. e Superposition of the EGF (blue)- and EREG (yellow)-bound EGFR/HER2 structures.

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Cryo-EM Sample Prep

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a Cryo-EM map of the interface between Domain IIs of EGFR and HER2 shown in two views. b Structure of the EGFR/HER2 interface in ribbon presentation. EGFR is colored in green and HER2 is in magenta. The BSAs of different regions are indicated. c – f Interaction details between EGFR and HER2 as indicated in the insets of b . Residues involved in their interaction are shown with side chains. Black dashed lines represent hydrogen bond or salt bridge interactions (< 4.5 Å). g B-factor distribution of the DAs in the EGFR–EGF/HER2 structure. h Comparison of the structures of DAs in different HER dimers. From left to right: EGFR–EGF (PDB code: 1IVO), dEGFR–Spitz (PDB code: 3LTG), EGFR–EREG (PDB code: 5WB7), EGFR–EGF/HER2 (this study), and HER2/HER3–NRG-1β (PDB code: 7MN5). The DAs of the symmetric EGFR dimer exhibit the same conformation. For asymmetric dimers, the DAs of the unbent subunit pack closely with its counterpart, mimicking that of the symmetric EGFR dimer, whereas those of the bent subunit display various structures. The distances between DAs of the bent subunits and their partners are indicated.

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a Pull-down assay to verify the significance of DAs for EGF-induced EGFR dimerization. The mCherry-tagged EGFR was associated with the mCherry-nanobody (Nb) resin, and GFP-tagged EGFR was used as input for this assay. b , c Pull-down assay to verify the significance of DAs for EGF- ( b ) or EREG-induced ( c ) EGFR/HER2 interaction. The mCherry-tagged HER2 was associated with the mCherry-Nb resin, and GFP-tagged EGFR was used as input for this assay. d Detection of the phosphorylation levels of EGFR, HER2, and AKT upon EGF stimulation. Different EGFR and HER2 variants were transfected to the EGFR-knockout SUM159 cells. e Pull-down assay to verify the significance of DAs for EREG-induced EGFR dimerization.

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Pull Down Assay, Transfection, Knock-Out

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a Live-cell single-molecule imaging and tracking of endogenous (en) EGFR-Halo and HER2-Halo molecules at the plasma membrane of SUM159 cells genome-edited for EGFR-Halo/HER2-mEGFP or HER2-Halo/EGFR-mEGFP (labeled by JFX 650 -HaloTag ligand). Shown are representative single frames and tracking traces of time-lapse series acquired in the cells treated without or with EGF (100 ng/mL) by TIRF microscopy. b MSD-Δt plots (left two panels) and diffusion coefficients (middle panel) of EGFR-Halo tracks ( n = 115, 109, and 110 cells from 4 independent experiments) and HER2-Halo tracks ( n = 117, 119, 120 cells from four independent experiments) from genome-edited SUM159 cells treated with 0, 10, or 100 ng/mL EGF and imaged by TIRF microscopy. The right panel shows fractions of tracks classified as mobile, confined, or immobile ( n = 4 independent experiments). c SK-BR-3 cells genome-edited for EGFR-Halo or HER-Halo were labeled by JFX 650 -HaloTag ligand, treated with 0, 10, or 100 ng/mL EGF, and imaged by TIRF microscopy. MSD-Δt plots (left two panels) and diffusion coefficients (middle panel) of EGFR-Halo tracks ( n = 150, 156, and 158 cells from 4 independent experiments) or HER-Halo tracks ( n = 152, 149, 153 cells from 4 independent experiments), and fractions of tracks classified as mobile, confined, or immobile (right panel, n = 4 independent experiments) are shown. d SUM159 cells genome-edited for both EGFR-SNAP and HER2-Halo were labeled by JFX 650 -SNAP-tag ligand and JFX 549 -HaloTag ligand, treated without or with EGF, and then imaged by TIRF microscopy. The representative single frame and tracking traces (co-localized trajectories are highlighted in blue) of the time-lapse series of a cell treated with 100 ng/mL EGF were shown on the left. Individual 3D trajectories (top) and distances (bottom) between EGFR-SNAP and HER2-Halo as a function of time are shown in the middle panels. The relative fractions of HER2 tracks that interact with EGFR in cells treated with 0, 10, or 100 ng/mL EGF are shown on the right ( n = 73, 77, and 77 cells from 2 independent experiments). e SUM159 cells genome-edited for EGFR-SNAP were transiently expressed with HaloTag-tagged CD86, HER2, HER2-GS, EGFR, and EGFR-GS, labeled by JFX 650 -SNAP-tag ligand and JFX 549 -HaloTag ligand, and then imaged by TIRF microscopy. Shown are the relative fractions of tracks in Halo channels that interact with the SNAP-tagged endogenous EGFR ( n = 36–40 cells from 2 independent experiments). Scale bars, 5 μm. Error bars show means ± SD except for the MSD-Δt plots (means ± 95% confidence interval).

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Imaging, Membrane, Labeling, Microscopy, Diffusion-based Assay

Journal: Cell Discovery

Article Title: Structure and dynamics of the EGFR/HER2 heterodimer

doi: 10.1038/s41421-023-00523-5

Figure Lengend Snippet: a SK-BR-3 cells genome-edited for EGFR-mEGFP or HER2-mEGFP were imaged by spinning-disk confocal microscopy. Shown are images of the cells in the middle planes at indicated times after EGF (100 ng/mL) treatment. Scale bars, 10 μm. b SK-BR-3 cells genome-edited for EGFR-mEGFP or HER2-mEGFP stably expressing clathrin-mScarlet-I were imaged at the bottom surfaces by TIRF microscopy. Shown are the single frames before and 3 min after EGF treatment during the continuous time-lapse imaging. Boxed regions are enlarged and shown. Scale bars, 5 μm. c SK-BR-3 cells genome-edited for EGFR-mEGFP and stably expressing clathrin-mScarlet-I were treated with control siRNA or siRNA targeting HER2 (HER2-KD), and then imaged at the bottom surfaces by TIRF microscopy. EGF was added at 120 s of the time-lapse imaging. The relative numbers of fluorescence spots of EGFR-mEGFP that appeared at the plasma membrane (left panel) and the relative enrichment of EGFR-mEGFP fluorescence in clathrin-coated structures (CCSs, right panel) during EGF stimulation are shown ( n = 23 and 19 cells). d SK-BR-3 cells genome-edited for EGFR-mEGFP and stably expressing clathrin-mScarlet-I were treated with control siRNA or siRNA targeting HER2 (HER2-KD). The cells treated with siRNA targeting HER2 were transiently transfected with the siRNA-resistant wild-type HER2 or the HER2-GS mutant and then imaged at the bottom surfaces by TIRF microscopy. EGF was added at 120 s of the time-lapse imaging. The relative numbers of fluorescence spots of EGFR-mEGFP that appeared at the plasma membrane (left panel, n = 49, 50, 29, and 30 cells) and the relative enrichment of EGFR-mEGFP fluorescence in CCSs (right panel, n = 43, 49, 28, and 30 cells) during EGF stimulation are shown. Error bars show means ± SEM.

Article Snippet: Genome-edited SUM159 cells expressing both EGFR-SNAP and HER2-Halo were cultured overnight in the pretreated confocal dishes (Cellvis).

Techniques: Confocal Microscopy, Stable Transfection, Expressing, Microscopy, Imaging, Fluorescence, Membrane, Transfection, Mutagenesis

Journal: Breast Cancer Research : BCR

Article Title: MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

doi: 10.1186/s13058-019-1104-5

Figure Lengend Snippet: a Representative pictures of single mammospheres of the cell lines MCF7, BT474, and SUM159 used for profiling the whole miRNome. b–c Heat map and corresponding tables with fold changes of the top 10 down- and upregulated miRNAs in mammospheres compared to adherent growing cells. d A significantly lower expression level (4.6-fold downregulation) for miR-1287-5p was found in cancer tissue compared to corresponding normal tissue. e Low levels of miR-1287 were a negative prognostic factor for survival in an independent external large cohort of 1262 breast cancer patients (HR = hazard ratio)

Article Snippet: 1 × 10 6 SUM159 cells were injected subcutanously in mammary fad-pad in a volume of 20 μl to NMRI:nu/nu mice (Janvier Labs, Paris, France).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

doi: 10.1186/s13058-019-1104-5

Figure Lengend Snippet: a–f Effects of transient overexpression or inhibition of miR-1287-5p on cellular growth using colony formation unit (CFU) assay in three different triple negative cell lines. Bar chart graphs represent relative number of colonies in percentage compared to control transfected cells ( n = 3) ( a , c, and e ), representative pictures are shown ( b , d, and f ). Mir-1287-5p mimics led to a significant decrease in cellular growth, while miR-1287-5p inhibitor exerted the opposite effect. g–h Effect of stable overexpression of miR-1287-5p on cellular growth measured by WST-1 proliferation assay and ( i–l ) CFU assay in two different triple negative breast cancer cell lines. Stable overexpression of miR-1287-5p led to a significantly reduced cellular growth in the cell lines SUM159 and MDA-MB-231. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: 1 × 10 6 SUM159 cells were injected subcutanously in mammary fad-pad in a volume of 20 μl to NMRI:nu/nu mice (Janvier Labs, Paris, France).

Techniques: Over Expression, Inhibition, Colony-forming Unit Assay, Control, Transfection, Proliferation Assay

Journal: Breast Cancer Research : BCR

Article Title: MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

doi: 10.1186/s13058-019-1104-5

Figure Lengend Snippet: In vivo xenograft experiments of stable mature form of miR-1287-5p overexpressing SUM159 cells. MiR-1287-5p overexpressing cells were injected in the left mammary fat pad of nude mice compared to control SUM159 cells which were injected into the right site. a–c Cells with miR-1287-5p overexpression developed significantly smaller tumors compared to control cells. ( d ) Bar chart of histomorphometric measurements of the largest tumor area detected in HE-staining revealed significantly smaller tumor area in SUM159 miR-1287-5p overexpressing cells * p < 0.05, *** p < 0.001. ( e - f ) Representative histological pictures of control cells and miR-1287-5p overexpressing SUM159 (HE staining, × 4 magnification, inserts with x40 magnification)

Article Snippet: 1 × 10 6 SUM159 cells were injected subcutanously in mammary fad-pad in a volume of 20 μl to NMRI:nu/nu mice (Janvier Labs, Paris, France).

Techniques: In Vivo, Injection, Control, Over Expression, Staining

Journal: Breast Cancer Research : BCR

Article Title: MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

doi: 10.1186/s13058-019-1104-5

Figure Lengend Snippet: Target identification of miR-1287-5p in triple negative breast cancer cells. a qRT-PCR confirmed a significant downregulation of the PIK3CB mRNA in all four tested triple negative BC cell lines after forced ectopic miR-1287-5p overexpression after 48 h of transfection. b Western blot analysis confirmed a significant downregulation of PIK3CB on protein level after transient transfection of miR-1287-5p in all tested cell lines (SUM159, BT549, MDA-MB-231, and MDA-MB-468) after 48 h of transfection. Relative quantification (numbers above the lanes) of protein lanes was performed using ImageJ. c Predicted miR-1287-5p interaction site within 3′ untranslated region of PIK3CB mRNA. Two PIK3CB constructs were generated as indicated (WT = miR-1287 wild-type interacting site and MT = mutated interacting site). d Luciferase activity after co-transfection of the PIK3CB wild-type or mutated constructs and control/miR-1287-5p mimetic in HEK cells. Three independent biological experiments were performed, and the means and standard deviations are shown. e High-PIK3CB expression is associated with poor clinical outcome in 1005 BC patients of a TCGA dataset.* p < 0.05

Article Snippet: 1 × 10 6 SUM159 cells were injected subcutanously in mammary fad-pad in a volume of 20 μl to NMRI:nu/nu mice (Janvier Labs, Paris, France).

Techniques: Drug discovery, Quantitative RT-PCR, Over Expression, Transfection, Western Blot, Quantitative Proteomics, Construct, Generated, Luciferase, Activity Assay, Cotransfection, Control, Expressing

Journal: Breast Cancer Research : BCR

Article Title: MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

doi: 10.1186/s13058-019-1104-5

Figure Lengend Snippet: a–c Clonogenic assay of the cell lines SUM159, MDA-MB-231, and BT549 after transient silencing of the putative miR-1287-5p target PIK3CB leads to a similar phenotype compared to miR-1287 overexpression in the cell lines. Cells develop less colonies after PIK3CB silencing ( a , b ) and PIK3CB silencing also leads to a G1 Phase Arrest ( c ) in all four cell lines. d–g SUM159 and BT549 cells treated with two different concentrations of PI3Kinase inhibitors in combination with control scrambled RNA (10 μM of Allstar negative control) or the miR-1287-5p mimics (10 μM of miR-1287-5p mimics) ( d , e ) CAL101 (Idelalisib) and f , g BYL719 (Alpelisib). Cells treated with miR-1287-5p mimic are more sensitive to CAL-101 and BYL719 treatment in both tested cell lines compared to cells treated with the scrambled control RNA. * p < 0.05

Article Snippet: 1 × 10 6 SUM159 cells were injected subcutanously in mammary fad-pad in a volume of 20 μl to NMRI:nu/nu mice (Janvier Labs, Paris, France).

Techniques: Clonogenic Assay, Over Expression, Control, Negative Control

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: A phenolic-enriched crude olive oil extract inhibits the mammosphere-initiating capacity of BC CSC-like states. ( A ) Three-dimensional map of phenolic compound separation obtained by HPLC-ESI-TOF in a crude EVOO-PE obtained via isolation protocols described in , available at Carcinogenesis Online. ( B ) Representative ALDEFLUOR ® assay to identify SUM-159 cells with high ALDH activity (ALDH + ) in the absence or presence of 10 μg/mL of the crude EVOO-PE for 3 days. The ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as negative control. Monolayer cultures were fed with the crude EVOO-PE every other day. Results are representative of two technical replicates per n ; n = 3 biological replicates. MSFE is expressed as percentages means (columns) ± SD (bars); three technical replicates per n ; n = 3 biological replicates. MTT uptake-based measurement of cell viability is expressed as percentages uptake (OD 570 ) relative to untreated control cells (=100% cell viability). The results are expressed as percentages means (columns) ± SD (bars); three technical replicates per n ; i = 3 biological replicates. ( C ) Figure shows representative light microscope representations (×20 magnification) of mammospheres formed by HMLER ShControl and HMLER ShEcad cells growing in sphere medium for 7 days in the absence or presence of graded concentrations of EVOO-PE. MSFE and MTT calculations were performed as described for SUM-159 cells in the left panels; three technical replicates per n ; n = 3 biological replicates. (* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group).

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Isolation, Activity Assay, Negative Control, Light Microscopy

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: DOA specifically and potently decreases the mammosphere-initiating capacity of BC CSC-like states. ( A ) Representative ALDEFLUOR ® assay to identify SUM-159 cells with high ALDH activity (ALDH + ) in the absence or presence of 20 μmol/L DOA for 3 days. The ALDH inhibitor DEAB was used as negative control. Monolayer cultures were fed with the DOA every other day. Results are representative of two technical replicates per n ; n = 3 biological replicates. ( B ) Figure shows representative light microscope representations (×20 magnifications) of mammospheres formed by HMLER ShEcad cells growing in sphere medium for 7 days in the absence or presence of graded concentrations of DOA. MSFE and MTT calculations were performed as described in ; three technical replicates per n ; n = 3 biological replicates. (* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group; n.s. not statistically significant). ( C ) Anoikis-resistant cells obtained as described in , available at Carcinogenesis Online were cultured in DOA-free mammosphere medium for 7 days, and MSFE were calculated following the same procedure as that described in and 2; three technical replicates per n ; n = 2 biological replicates.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Activity Assay, Negative Control, Light Microscopy, Cell Culture

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: DOA blocks tumor-initiating capacity of CSC-like cells in vivo . ( A ) Tumor growth rate was calculated by measuring volumes along several weeks after injection of DOA-pretreated of untreated control cells. Shown are the mean volumes (±SD) for at least 9 weeks. Tumor-free survival in mice bearing SUM-159 xenografts with volumes ≥10 and ≥50 mm 3 is shown as a function of time. [* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group; n.s. not statistically significant]. ( B ) Mammosphere-initiating cells were isolated and exposed to graded concentrations of DOA for 2 h. Viable single cell suspensions (1 × 10 4 cells) were orthotopically injected into the mammary fat pads of SCID/Beige mice, and tumor growth was monitored for at least 4 months. Tumor-free survival in mice bearing orthotopically implanted SUM-159 CSC-like cells is shown as a function of time.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: In Vivo, Injection, Isolation

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: ( A ) DOA synergistically interacts with mTOR and DNMT inhibitors. Shown are representative microphotographs of PM arrays (microplates PM-M11–PM-M14) from two biological replicates. The different interactions were defined as described in , available at Carcinogenesis Online. ( B ) DOA decreases mTOR and DNMT activities in cultured cells. Left. Representative western blotting analyses of phospho-p70 S6 Kinase (Thr389)/phospho-p85 S6 Kinase (Thr412) in SUM-159 cells cultured in the absence of presence of DOA or rapamycin for up to 6 h, as specified. Right. Nuclear extracts of SUM-159 were exposed to 20 μmol/L DOA for 2 h before assessing DNMT activity using the DNMT activity/Inhibition Assay Kit of Active Motif as per manufacturer’s instructions. The results are expressed as percentages of the means (columns) ± SD (bars); three technical replicates per n ; n = 2 biological replicates. (** P < 0.001 versus DNMT activity in untreated nuclear extracts). ( C ) DOA binds the ATP-binding pocket in mTOR kinase domain and inhibits mTOR kinase activity. Overall structures and views of the interaction between DOA (red) and well-known TORKinhibs with the ATP-dependent catalytic pocket of mTOR (PDB code 4JT6). Figure shows in sticks all the interaction residues involved in the binding of DOA/TORKinhibs using PLIP. Hydrogen bond interactions are represented by orange dashed lines; salt bridges are represented by yellow dashed lines and charge centers by yellow spheres; Cation-π interactions are represented by blue dashed lines and white spheres for the center of the aromatic ring. ( D ) DOA directly inhibits the ATP-dependent catalytic activity of mTOR. A dose-response curve of ATP-dependent mTOR kinase activity was created by plotting FRET signal of the Z′-LYTE Kinase assay as the function of DOA concentration.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Cell Culture, Western Blot, Activity Assay, Inhibition, Binding Assay, Kinase Assay, Concentration Assay

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After mechanical preconditioning, SUM159, SKBR3 and MS-SKBR3.1 cells expressing iRFP-LifeAct were trypsizined from their hydrogels and plated onto No. 1.5 glass MatTek dishes which had been pre-treated overnight with DMEM + 10% FBS, and then incubated for 10 hours to ensure maximal spreading before analysis (verified by size equilibrium).

Techniques: Recombinant, Membrane, Enzyme-linked Immunosorbent Assay, Software