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  • 99
    ATCC mda mb231 cells
    Expression of endogenous AKIP1 in various cells. 293, HeLa, MCF7, and <t>MDA-MB231</t> cells were cultured and lysed for Western blotting using polyclonal antibody against AKIP1. Note the different expression levels of endogenous AKIP1 in these cell lines. The protein expression levels of α-tubulin indicate that the same amounts of cell lysates were loaded.
    Mda Mb231 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mda mb231 cells
    Acetylation of H2A.Z at the p21 promoter is necessary for its transcription. a) Schematic representation of the p21 promoter region showing PCR amplified fragments (1–4). b) Binding of H2A.Z to the p21 promoter in <t>MDA-MB231</t> cells. c) mRNA expression of H2AFZ (left) and p21 (right) in MDA-MB231 cells transfected with a smartpool siH2A.Z for 72h. d) H2A.Z and polymerase II (pol II) binding to the p21 TSS (fragment #4) in cells transfected with siH2A.Z or scramble siRNAs. e) H2A.Z binding to the p21 TSS (fragment #4) in cells treated with TSA (50 ng/ml) for 48 h. f–g) acetylated H2A.Z, polymerase II (pol II) (f) and acetylated H3K9 (g) binding to the p21 TSS (fragment #4) in cells treated with TSA. h-i) acetylated H2A.Z (h) and H3K9 (i) amount at p21 TSS in cells transfected with siH2A.Z or scramble siRNA.
    Mda Mb231 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Selleck Chemicals mda mb 231 cells
    Loss of REST leads to increased IRS1 protein levels. (A) Western blot images showing REST and IRS1 expression in MCF7 (ER + ), <t>MDA-MB-231</t> (ER − ), and ZR751 (ER + ) cells. (B) Quantifications of relative protein expression depicted in a graph (for shRNA
    Mda Mb 231 Cells, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 90/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    DSMZ mda mb 231 cells
    Experimental setup and migration analysis. (A) Technical drawing of the μ -Slide Chemotaxis showing the three adjacent chambers for parallel experiments. Two reservoirs are connected by a central observation area (height: 70 μ m, width: 1 mm). Single cells are embedded in a 3D matrix inside the observation area. A time-stable gradient can be established by filling the reservoirs with different concentrations of chemoattractant (C 0 and C 100 ). (B) Representative image of the observation area with <t>MDA-MB-231</t> cells embedded in a collagen gel with an overlay of the manually tracked cell trajectories at time point 24 h. Scale bar represents 500 μ m. (C) Each experimental setup includes three conditions: the experimental condition offering a concentration gradient (+/-) and the two controls with chemoattractant being present in the entire system (+/+) or without chemoattractant (-/-). (D) Analysis of chemotaxis by forward migration indices in direction of the gradient (FMI II ) and perpendicular to the gradient (FMI┴). FMI II values are high for directional migration and close to zero for random migration.
    Mda Mb 231 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 96/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boster Bio mda mb 231 cells
    Influence of TGF-β1 on TNBC cell signal transduction pathways. (A) <t>MDA-MB-231</t> cells were treated with 5 ng/ml TGF-β1 and then analyzed by western blot analysis. Smad2 protein and phosphorylation of Smad2 protein, P38 protein and phosphorylation
    Mda Mb 231 Cells, supplied by Boster Bio, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Caliper Life Sciences mda mb 231 cells
    Macrophages associate with developing xenograft vessels. (A-G) Still images showing an <t>MDA-MB-231</t> xenograft (A-D, Movie 3 ) or a B16-F1 xenograft (E-G, Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). Individual macrophages associated with the angiogenic region (yellow dashed circle) are indicated with yellow arrowheads, while macrophages associated with the control region (cyan dashed circle) are indicated with cyan arrowheads. The tumour region is outlined by a white dashed line in A and E. (H) Schematic demonstrating the positioning of the 10-μm angiogenic region (dashed yellow outline) at the tip of the blood vessel (green) and the control 10-μm control region (dashed cyan outline). (I) Quantitation of the percentage of frames during which a macrophage was observed at either an angiogenic region or a control region in MDA-MB-231 and B16-F1 xenografts, n =3. (J) Mean number of macrophages observed during each frame, in either the angiogenic region or the control region, in MDA-MB-231 and B16 F1 xenografts, n =3. (K-R) Still images showing a B16-F1 xenograft ( Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). An individual macrophage (yellow arrowhead) is tracked for 1 h 10 min during (K-N) and after (O-R) associating with the distal tip of a growing vessel (indicated by a cyan arrowhead). (S,T) Macrophage migration tracks of 12 macrophages during their period of contact with the tip of a growing vessel (S) and of the same macrophages once they leave the vessel tip (T). Each macrophage is depicted with the same colour in both S and T and they were tracked for identical periods of time during contact and post-contact. (U) Quantitation of macrophage migration speed during and after the period of contact with the tip of a growing vessel, n =12. Error bars represent s.d. * P
    Mda Mb 231 Cells, supplied by Caliper Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem mda mb 231 cells
    Role of MEK/ERK signaling pathway in migration induced by refp17 and vp17s. <t>MDA-MB</t> 231 cells were serum starved for 24 h in the presence or absence of the PI3K/Akt inhibitor LY294002 (20 μM), the Jak/STAT inhibitor AG-490 (20 μM), or the MEK/ERK1/2 inhibitor PD98059 (10 μM). a Confluent cell monolayers were serum starved for 24 h and then scratched with a 200 μl pipette tip. Cells were then incubated for 6 h in the absence (NT) or in the presence of 10 ng/ml of refp17 or vp17s. Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (*** p
    Mda Mb 231 Cells, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher mda mb 231 cells
    Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and <t>MDA-MB-231</t> cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.
    Mda Mb 231 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 9151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Caliper Life Sciences mda mb 231 d3h2 ln mb231 cells
    Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and <t>MDA-MB-231</t> cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.
    Mda Mb 231 D3h2 Ln Mb231 Cells, supplied by Caliper Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Essen Bioscience nuclight rfp mda mb 231 cells
    Olaparib attenuates PD-1 binding and T-cell-mediated cell death in TNBC cells (A) FACS analysis of cell surface PD-1 binding of <t>MDA-MB-231</t> cells treated with 10 μM olaparib or 10 nM talazoparib for 24 hours. (B) FACS analysis of PARP1 knockdown (K/D), PARP1 knockout (K/O), and parental MDA-MB-231 cells. (C) SUM149 cells were treated with the indicated concentrations of olaparib for 10 days. (D) MDA-MB-231 cells expressing nuclear <t>RFP</t> protein were first treated with or without olaparib (10 μM) for 3 hours and then co-cultured with or without activated peripheral blood mononuclear cells (PBMCs). Left, quantitation showing the number of live cells per well, counting the number of red fluorescent objects, normalized to that at the zero time point. Right, the percent of T cell-meditated tumor cell killing observed at 72 hours in activated PBMC co-culture with control or olaparib-treated cells (normalized to co-culture without PBMCs). (E) Left, representative merged images showing red fluorescent (nuclear restricted RFP), and green fluorescent (Caspase 3/7 substrate) objects in MDA-MB-231 cells co-cultured with activated PBMCs at 0 and 72 hours. Images were taken using the IncuCyte Zoom microscope. Right, quantitation showing the number of live cells following treatment with olaparib (10 μM), PD-L1 antibody (PD-L1 Ab; 10 μg/ml), or the combination co-cultured with activated PBMCs for 72 hours. The number of live cells (red fluorescent objects) were counted and normalized to that at the zero time point. * P
    Nuclight Rfp Mda Mb 231 Cells, supplied by Essen Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Obio Technology Corp Ltd cell culture mda mb 231 cells
    Flow cytometry analysis of apoptosis in <t>MDA-MB-231</t> cells. a Representative images of flow cytometry. b Apoptotic rates in cells treated with the stated concentrations of BPTS. ** P
    Cell Culture Mda Mb 231 Cells, supplied by Obio Technology Corp Ltd, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of endogenous AKIP1 in various cells. 293, HeLa, MCF7, and MDA-MB231 cells were cultured and lysed for Western blotting using polyclonal antibody against AKIP1. Note the different expression levels of endogenous AKIP1 in these cell lines. The protein expression levels of α-tubulin indicate that the same amounts of cell lysates were loaded.

    Journal: The Journal of Biological Chemistry

    Article Title: A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-?B Signaling

    doi: 10.1074/jbc.M110.116566

    Figure Lengend Snippet: Expression of endogenous AKIP1 in various cells. 293, HeLa, MCF7, and MDA-MB231 cells were cultured and lysed for Western blotting using polyclonal antibody against AKIP1. Note the different expression levels of endogenous AKIP1 in these cell lines. The protein expression levels of α-tubulin indicate that the same amounts of cell lysates were loaded.

    Article Snippet: Cell lysates from 293, HeLa, MCF7, and MDA-MB231 cells were collected, and Western blotting assay was performed by using polyclonal antibody raised against AKIP1 (kindly provided by Professor S. Taylor) ( ).

    Techniques: Expressing, Multiple Displacement Amplification, Cell Culture, Western Blot

    Cell cycle analysis in MDA-MB231 cells at 48 h after treatment with the Xanthoria parietina extract (1.5 mg/mL) or parietin at the indicated concentrations.

    Journal: International Journal of Molecular Sciences

    Article Title: Antiproliferative, Antibacterial and Antifungal Activity of the Lichen Xanthoria parietina and Its Secondary Metabolite Parietin

    doi: 10.3390/ijms16047861

    Figure Lengend Snippet: Cell cycle analysis in MDA-MB231 cells at 48 h after treatment with the Xanthoria parietina extract (1.5 mg/mL) or parietin at the indicated concentrations.

    Article Snippet: Cancer Cell Lines and Materials MCF-7 and MDA-MB231 cell lines were obtained from ATCC and routinely cultured.

    Techniques: Cell Cycle Assay, Multiple Displacement Amplification

    Morphological analysis of proliferation arrest in MDA-MB231 breast cancer cells at 24 and 48 h after treatment with 1.5 mg/mL of the Xanthoria parietina extract (AE) or parietin (P) at the indicated concentrations (50-100-200 µM). Control (CTR), scale bar: 71 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Antiproliferative, Antibacterial and Antifungal Activity of the Lichen Xanthoria parietina and Its Secondary Metabolite Parietin

    doi: 10.3390/ijms16047861

    Figure Lengend Snippet: Morphological analysis of proliferation arrest in MDA-MB231 breast cancer cells at 24 and 48 h after treatment with 1.5 mg/mL of the Xanthoria parietina extract (AE) or parietin (P) at the indicated concentrations (50-100-200 µM). Control (CTR), scale bar: 71 μm.

    Article Snippet: Cancer Cell Lines and Materials MCF-7 and MDA-MB231 cell lines were obtained from ATCC and routinely cultured.

    Techniques: Multiple Displacement Amplification

    Specific association of MBD2 to the methylated promoter region of pS2 gene. Detail of the pS2 gene region analyzed (from nt positions −464 to +314). CpG sites are represented by circles. The black line represents the position of the fragment amplified by dose-dependant and quantitative PCR after ChIP. MBD proteins binding to the methylated region of the pS2 promoter (from nt positions −11 to +292) was analyzed by ChIP in MDA MB231, HeLa and MCF7 cells. Cross-linked chromatin was immunoprecipitated using rabbit polyclonal anti-MBD2, anti-MeCP2 and anti-MBD1 antibodies. Purified DNAs from the input, unbound, bound or IgG fractions were quantified and an equal quantity of each fraction (0.5 ng) of this DNA was amplified by dose-dependent ( A ) or quantitative ( B ) PCR. ( A ) Representative experiments of MBD occupancy in the pS2 promoter are shown. ( B ) Relative amounts of immunoprecipitaded pS2 promoter to the input fraction measured by quantitative PCR. Each bar represents the mean ± standard deviation of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

    doi: 10.1371/journal.pone.0009665

    Figure Lengend Snippet: Specific association of MBD2 to the methylated promoter region of pS2 gene. Detail of the pS2 gene region analyzed (from nt positions −464 to +314). CpG sites are represented by circles. The black line represents the position of the fragment amplified by dose-dependant and quantitative PCR after ChIP. MBD proteins binding to the methylated region of the pS2 promoter (from nt positions −11 to +292) was analyzed by ChIP in MDA MB231, HeLa and MCF7 cells. Cross-linked chromatin was immunoprecipitated using rabbit polyclonal anti-MBD2, anti-MeCP2 and anti-MBD1 antibodies. Purified DNAs from the input, unbound, bound or IgG fractions were quantified and an equal quantity of each fraction (0.5 ng) of this DNA was amplified by dose-dependent ( A ) or quantitative ( B ) PCR. ( A ) Representative experiments of MBD occupancy in the pS2 promoter are shown. ( B ) Relative amounts of immunoprecipitaded pS2 promoter to the input fraction measured by quantitative PCR. Each bar represents the mean ± standard deviation of at least three independent experiments.

    Article Snippet: Cell culture MDA MB231, MCF7, and HeLa cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

    Techniques: Methylation, Amplification, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, Purification, Polymerase Chain Reaction, Standard Deviation

    pS2 gene expression and DNA methylation patterns in MCF7, HeLa and MDA MB231 cells. ( A ) The expression of endogenous pS2 gene in MCF7, HeLa and MDA MB231 cell lines. pS2 mRNA levels were monitored by relative RT-PCR. Briefly, pS2 transcripts were simultaneously amplified with β-actin transcripts as a loading control and expression standard. ( B ) Methylation patterns at CpG sites of pS2 5′ flanking sequence from nt positions −464 to +314 in MCF7, HeLa, and MDA MB231 cell lines. A schematic representation of the human pS2 gene is shown. The transcription start site is indicated by a black arrow. Black box, AP1 site; dark-grey box, Estrogen-Responsive Element (ERE); light-grey box, TATA-box; hatched boxes, pS2 exons. The studied region (from nt positions −464 to +314) is presented on an expanded scale. This region contains 20 CpG sites, represented by white circles. The bisulphite-sequencing status of this 5′ pS2 region in MCF7, HeLa and MDA MB231 cells (number of analyzed clones, n = 10) is represented. Each line corresponds to a single DNA template molecule. Black and open circles represent methylated and unmethylated CpGs, respectively.

    Journal: PLoS ONE

    Article Title: A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

    doi: 10.1371/journal.pone.0009665

    Figure Lengend Snippet: pS2 gene expression and DNA methylation patterns in MCF7, HeLa and MDA MB231 cells. ( A ) The expression of endogenous pS2 gene in MCF7, HeLa and MDA MB231 cell lines. pS2 mRNA levels were monitored by relative RT-PCR. Briefly, pS2 transcripts were simultaneously amplified with β-actin transcripts as a loading control and expression standard. ( B ) Methylation patterns at CpG sites of pS2 5′ flanking sequence from nt positions −464 to +314 in MCF7, HeLa, and MDA MB231 cell lines. A schematic representation of the human pS2 gene is shown. The transcription start site is indicated by a black arrow. Black box, AP1 site; dark-grey box, Estrogen-Responsive Element (ERE); light-grey box, TATA-box; hatched boxes, pS2 exons. The studied region (from nt positions −464 to +314) is presented on an expanded scale. This region contains 20 CpG sites, represented by white circles. The bisulphite-sequencing status of this 5′ pS2 region in MCF7, HeLa and MDA MB231 cells (number of analyzed clones, n = 10) is represented. Each line corresponds to a single DNA template molecule. Black and open circles represent methylated and unmethylated CpGs, respectively.

    Article Snippet: Cell culture MDA MB231, MCF7, and HeLa cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

    Techniques: Expressing, DNA Methylation Assay, Multiple Displacement Amplification, Reverse Transcription Polymerase Chain Reaction, Amplification, Methylation, Sequencing, Bisulfite Sequencing, Clone Assay

    ERα only associates hypomethylated ERE region of pS2 . Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2 . The position of the “ pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.

    Journal: PLoS ONE

    Article Title: A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

    doi: 10.1371/journal.pone.0009665

    Figure Lengend Snippet: ERα only associates hypomethylated ERE region of pS2 . Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2 . The position of the “ pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.

    Article Snippet: Cell culture MDA MB231, MCF7, and HeLa cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

    Techniques: Chromatin Immunoprecipitation, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    Characterization of ELP variants. a Schematic representation of the amino acid sequences of the designed variants (E 28 , Tat-E 28 , Tat-A 1 E 28 , and Tat-A 4 V 48 ). The positively charge amino acids present in ELPs induced complexation with negatively charged nucleic acids to form nanoparticle like structures. b Turbidity profiles of ELPs determined by measuring O.D. values at 350 nm while increasing temperature at 1 °C/min. Cell binding activities of the ELPs (0.3125 µM) were measured by flow cytometry after incubating MDA MB231 ( c ) or 4T1 ( d ) cells with the respective polypeptides labeled with Alexa-488 at 4 °C. Results are presented as means ± SDs (n = 6). ***P

    Journal: Journal of Nanobiotechnology

    Article Title: Development of elastin-like polypeptide for targeted specific gene delivery in vivo

    doi: 10.1186/s12951-020-0574-z

    Figure Lengend Snippet: Characterization of ELP variants. a Schematic representation of the amino acid sequences of the designed variants (E 28 , Tat-E 28 , Tat-A 1 E 28 , and Tat-A 4 V 48 ). The positively charge amino acids present in ELPs induced complexation with negatively charged nucleic acids to form nanoparticle like structures. b Turbidity profiles of ELPs determined by measuring O.D. values at 350 nm while increasing temperature at 1 °C/min. Cell binding activities of the ELPs (0.3125 µM) were measured by flow cytometry after incubating MDA MB231 ( c ) or 4T1 ( d ) cells with the respective polypeptides labeled with Alexa-488 at 4 °C. Results are presented as means ± SDs (n = 6). ***P

    Article Snippet: Cell culture MDA-MB231 cells (a human breast cancer cell line) and 4T1-luc cells (a murine breast cancer cell line) were obtained from American Type Culture Collection (ATCC).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Labeling

    Sizes and structural analysis of siRNA/ELPs complexes and intracellular trafficking. a TEM images of nanoparticle-like structures formed by siRNA/ELPs complexes at room temperature. Scale bar = 100 nm. b In order to determine subcellular localizations and siRNA release, MDA MB231 cells were stained with anti-EEA1 (early endosome antigen) after incubating them with siRNA/ELP complexes for 1 h at 37 °C. The cellular localizations of complexes in early endosomes were observed by confocal microscopy. Representative confocal microscopic images of five experiments. Blue, nuclei stained with Hoechst; Green, siRNA/ELP variant complexes; Red, Anti-EEA1. Scale bar, 10 µm

    Journal: Journal of Nanobiotechnology

    Article Title: Development of elastin-like polypeptide for targeted specific gene delivery in vivo

    doi: 10.1186/s12951-020-0574-z

    Figure Lengend Snippet: Sizes and structural analysis of siRNA/ELPs complexes and intracellular trafficking. a TEM images of nanoparticle-like structures formed by siRNA/ELPs complexes at room temperature. Scale bar = 100 nm. b In order to determine subcellular localizations and siRNA release, MDA MB231 cells were stained with anti-EEA1 (early endosome antigen) after incubating them with siRNA/ELP complexes for 1 h at 37 °C. The cellular localizations of complexes in early endosomes were observed by confocal microscopy. Representative confocal microscopic images of five experiments. Blue, nuclei stained with Hoechst; Green, siRNA/ELP variant complexes; Red, Anti-EEA1. Scale bar, 10 µm

    Article Snippet: Cell culture MDA-MB231 cells (a human breast cancer cell line) and 4T1-luc cells (a murine breast cancer cell line) were obtained from American Type Culture Collection (ATCC).

    Techniques: Transmission Electron Microscopy, Multiple Displacement Amplification, Staining, Confocal Microscopy, Variant Assay

    RFP14 specifically inhibits RAB25-dependent migration in cancer cells. a , b Representative time-dependent scratch-wound cell migration assay images a and quantification b of mock vector and RAB25 -expressing HEY, MCF7, and MDA-MB231 cells. RAB25 expression results in significant enhancement (HEY, MCF7) or inhibition (MDA-MB231) of cell migration. These data are representative of triplicate biological replicates. c , d RFP 14 (10 μM) significantly impaired cell migration of RAB25 -expressing d , but not vector control c , HEY cells. e RFP14 (10 μM), but not RFP32, significantly impaired cell migration of RAB25 -expressing MCF7 cells. f Treatment of RAB25 -expressing MDA-MB231 cells with RFP14, conversely, significantly increased cell migration relative to vehicle while the inactive control RFP32 remained unchanged compared to vehicle control. Representative scratch wound images are shown for RFP14, RFP32, and vehicle (DMSO) treatment conditions with overlaid arrows highlighting distance between cell fronts. Images and data shown represent mean ± s.e.m. of triplicate biological replicates. * P

    Journal: Nature Communications

    Article Title: Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer

    doi: 10.1038/s41467-017-00888-8

    Figure Lengend Snippet: RFP14 specifically inhibits RAB25-dependent migration in cancer cells. a , b Representative time-dependent scratch-wound cell migration assay images a and quantification b of mock vector and RAB25 -expressing HEY, MCF7, and MDA-MB231 cells. RAB25 expression results in significant enhancement (HEY, MCF7) or inhibition (MDA-MB231) of cell migration. These data are representative of triplicate biological replicates. c , d RFP 14 (10 μM) significantly impaired cell migration of RAB25 -expressing d , but not vector control c , HEY cells. e RFP14 (10 μM), but not RFP32, significantly impaired cell migration of RAB25 -expressing MCF7 cells. f Treatment of RAB25 -expressing MDA-MB231 cells with RFP14, conversely, significantly increased cell migration relative to vehicle while the inactive control RFP32 remained unchanged compared to vehicle control. Representative scratch wound images are shown for RFP14, RFP32, and vehicle (DMSO) treatment conditions with overlaid arrows highlighting distance between cell fronts. Images and data shown represent mean ± s.e.m. of triplicate biological replicates. * P

    Article Snippet: Cell lines and reagents HEY, MCF7, MDA-MB231 cells lines were originally obtained from ATCC.

    Techniques: Migration, Cell Migration Assay, Plasmid Preparation, Expressing, Multiple Displacement Amplification, Inhibition

    RFP peptides alter RAB25-driven cell proliferation. a Western blot analysis of mock vector and RAB25 overexpressing HEY, MDA-MB231, and MCF7 cells. b Western blot analysis for endogenous RAB11a in HEY, MDA231 and MCF7 isogenic lines with or without RAB25 overexpression. c Left panel compares the viability of untreated HEY cells under low-serum condition in mock or RAB25 expressing lines while right panel shows the effects of treatment with DMSO or RFP14, 26, and 32 (15 μM) on the viability of HEY mock or RAB25 expressing cell lines at 48 h post-treatment. d , e shows corresponding results from MCF7 isogenics and MDA-MB231 isogenics, respectively. RAB25 expression results in enhancement (HEY, MCF7) or inhibition (MDA-MB231) of cell proliferation, which is reversed by RFP14, but not RFP32. Images and data shown represent mean ± s.e.m. of triplicate biological replicates. * P

    Journal: Nature Communications

    Article Title: Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer

    doi: 10.1038/s41467-017-00888-8

    Figure Lengend Snippet: RFP peptides alter RAB25-driven cell proliferation. a Western blot analysis of mock vector and RAB25 overexpressing HEY, MDA-MB231, and MCF7 cells. b Western blot analysis for endogenous RAB11a in HEY, MDA231 and MCF7 isogenic lines with or without RAB25 overexpression. c Left panel compares the viability of untreated HEY cells under low-serum condition in mock or RAB25 expressing lines while right panel shows the effects of treatment with DMSO or RFP14, 26, and 32 (15 μM) on the viability of HEY mock or RAB25 expressing cell lines at 48 h post-treatment. d , e shows corresponding results from MCF7 isogenics and MDA-MB231 isogenics, respectively. RAB25 expression results in enhancement (HEY, MCF7) or inhibition (MDA-MB231) of cell proliferation, which is reversed by RFP14, but not RFP32. Images and data shown represent mean ± s.e.m. of triplicate biological replicates. * P

    Article Snippet: Cell lines and reagents HEY, MCF7, MDA-MB231 cells lines were originally obtained from ATCC.

    Techniques: Western Blot, Plasmid Preparation, Multiple Displacement Amplification, Over Expression, Expressing, Inhibition

    Cytotoxic effect of hypoxia on MDA-MB-231 cells. The cytotoxicity values are 9.871 ± 0.553 % for hypoxic cells and 10.002 ± 0. 417 % for normoxic control cells, which indicated that hypoxic treatment had no significant cytotoxic effect on MDA-MB-231 cells ( P > 0.05)

    Journal: Medical Oncology (Northwood, London, England)

    Article Title: Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

    doi: 10.1007/s12032-016-0755-7

    Figure Lengend Snippet: Cytotoxic effect of hypoxia on MDA-MB-231 cells. The cytotoxicity values are 9.871 ± 0.553 % for hypoxic cells and 10.002 ± 0. 417 % for normoxic control cells, which indicated that hypoxic treatment had no significant cytotoxic effect on MDA-MB-231 cells ( P > 0.05)

    Article Snippet: Recovery and culture of MDA-MB-231 cells MDA-MB-231 cells were cultured in air using L-15 complete medium containing 10 % FBS, in accordance with the recommendations of the American Type Culture Collection ( www.atcc.org/ ).

    Techniques: Multiple Displacement Amplification

    Effect of hypoxia on expression of stem cell markers in MDA-MB-231 cells. a Normoxic control group; b hypoxic group; and c group comparison of percentage of cells expressing CD24 − CD44 + ESA + : it is significantly higher in the hypoxic group 3.60 ± 0.30 % compared with the normoxic group 1.33 ± 0.21 % ( P

    Journal: Medical Oncology (Northwood, London, England)

    Article Title: Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

    doi: 10.1007/s12032-016-0755-7

    Figure Lengend Snippet: Effect of hypoxia on expression of stem cell markers in MDA-MB-231 cells. a Normoxic control group; b hypoxic group; and c group comparison of percentage of cells expressing CD24 − CD44 + ESA + : it is significantly higher in the hypoxic group 3.60 ± 0.30 % compared with the normoxic group 1.33 ± 0.21 % ( P

    Article Snippet: Recovery and culture of MDA-MB-231 cells MDA-MB-231 cells were cultured in air using L-15 complete medium containing 10 % FBS, in accordance with the recommendations of the American Type Culture Collection ( www.atcc.org/ ).

    Techniques: Expressing, Multiple Displacement Amplification

    Effect of hypoxia on MDA-MB-231 cell apoptosis. a The effect of normoxia and hypoxia on MDA-MB-231 cell apoptosis, and b group comparison of apoptosis rate: normoxic control cells showed an apoptotic rate of 4.97 ± 0.42 %, compared with 0.97 ± 0.74 % in the hypoxic cells, indicating that the percentage of apoptotic cells is significantly reduced after 48 h of hypoxia ( P

    Journal: Medical Oncology (Northwood, London, England)

    Article Title: Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

    doi: 10.1007/s12032-016-0755-7

    Figure Lengend Snippet: Effect of hypoxia on MDA-MB-231 cell apoptosis. a The effect of normoxia and hypoxia on MDA-MB-231 cell apoptosis, and b group comparison of apoptosis rate: normoxic control cells showed an apoptotic rate of 4.97 ± 0.42 %, compared with 0.97 ± 0.74 % in the hypoxic cells, indicating that the percentage of apoptotic cells is significantly reduced after 48 h of hypoxia ( P

    Article Snippet: Recovery and culture of MDA-MB-231 cells MDA-MB-231 cells were cultured in air using L-15 complete medium containing 10 % FBS, in accordance with the recommendations of the American Type Culture Collection ( www.atcc.org/ ).

    Techniques: Multiple Displacement Amplification

    Effect of hypoxia on MDA-MB-231 cell proliferation. The growth curves of the two groups generally overlapped at 2, 6, 12, and 24 h, and the OD values are 0.560 ± 0.026 for control cells and 0.518 ± 0.014 for hypoxic cells ( P > 0.05)

    Journal: Medical Oncology (Northwood, London, England)

    Article Title: Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

    doi: 10.1007/s12032-016-0755-7

    Figure Lengend Snippet: Effect of hypoxia on MDA-MB-231 cell proliferation. The growth curves of the two groups generally overlapped at 2, 6, 12, and 24 h, and the OD values are 0.560 ± 0.026 for control cells and 0.518 ± 0.014 for hypoxic cells ( P > 0.05)

    Article Snippet: Recovery and culture of MDA-MB-231 cells MDA-MB-231 cells were cultured in air using L-15 complete medium containing 10 % FBS, in accordance with the recommendations of the American Type Culture Collection ( www.atcc.org/ ).

    Techniques: Multiple Displacement Amplification

    Effect of hypoxia on colony formation by MDA-MB-231 cells. a – c Show colony-formation rates in MDA-MB-231 cells in the 50- ( a ), 100- ( b ), and 200-cell groups ( c ), respectively

    Journal: Medical Oncology (Northwood, London, England)

    Article Title: Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

    doi: 10.1007/s12032-016-0755-7

    Figure Lengend Snippet: Effect of hypoxia on colony formation by MDA-MB-231 cells. a – c Show colony-formation rates in MDA-MB-231 cells in the 50- ( a ), 100- ( b ), and 200-cell groups ( c ), respectively

    Article Snippet: Recovery and culture of MDA-MB-231 cells MDA-MB-231 cells were cultured in air using L-15 complete medium containing 10 % FBS, in accordance with the recommendations of the American Type Culture Collection ( www.atcc.org/ ).

    Techniques: Multiple Displacement Amplification

    Metastasis of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors. 40 days after subcutaneous injection of either human MDA-MB-231/GFP cells, ASC/RFP cells or MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected. A. Visual macrometastatic lesions were observed in the liver, lungs only in mice co-injected with MDA-MB-231/GFP and ASC/RFP (arrows). B. H E sections of the liver and lungs of mice bearing MDA-MB-231/GFP+ASC/RFP tumors showing metastatic MDA-MB-231 cancer cells (insets). C. To quantitate micrometastases, DNA was prepared from mouse organs from two separate experiments (n = 10 mice/group) for detection of human chromosome 17 by real time RT-PCR. A significant increase in micrometastasis for MDA-MB-231/GFP+ASC/RFP tumors was detected in liver, lung and spleen. * P

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: Metastasis of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors. 40 days after subcutaneous injection of either human MDA-MB-231/GFP cells, ASC/RFP cells or MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected. A. Visual macrometastatic lesions were observed in the liver, lungs only in mice co-injected with MDA-MB-231/GFP and ASC/RFP (arrows). B. H E sections of the liver and lungs of mice bearing MDA-MB-231/GFP+ASC/RFP tumors showing metastatic MDA-MB-231 cancer cells (insets). C. To quantitate micrometastases, DNA was prepared from mouse organs from two separate experiments (n = 10 mice/group) for detection of human chromosome 17 by real time RT-PCR. A significant increase in micrometastasis for MDA-MB-231/GFP+ASC/RFP tumors was detected in liver, lung and spleen. * P

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Multiple Displacement Amplification, Injection, Mouse Assay, Quantitative RT-PCR

    Wound healing/scratch assay to measure effect of ASC conditioned medium on MDA-MB-231 migration

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: Wound healing/scratch assay to measure effect of ASC conditioned medium on MDA-MB-231 migration

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Wound Healing Assay, Multiple Displacement Amplification, Migration

    ASC effect on tumor markers. IHC was conducted as described in Materials and Methods .Paraffin-embedded tumor sections from MDA-MB-231/GFP and the MDA-MB-231/GFP+ASC/RFP groups were stained for vimentin, MMP9, IL-8, CD-31, and VEGF. Bright-field photomicrographs were taken and representative images are presented. Quantitative representation of the staining is indicated.

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: ASC effect on tumor markers. IHC was conducted as described in Materials and Methods .Paraffin-embedded tumor sections from MDA-MB-231/GFP and the MDA-MB-231/GFP+ASC/RFP groups were stained for vimentin, MMP9, IL-8, CD-31, and VEGF. Bright-field photomicrographs were taken and representative images are presented. Quantitative representation of the staining is indicated.

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Immunohistochemistry, Multiple Displacement Amplification, Staining

    The effect of ASCs on the growth of MDA-MB 231 cells. A. MDA-MB-231 were cultured in the bottom well of a Boyden Chamber and ASCs were cultured in the insert. Growth of MDA-MB-231 cells was assessed using the MTT assay. B. 2.5×10 4 ASCs were cultured in 6 well plates for 24 hrs. prior to addition of MDA-MB-231-GFP breast cancer cells at a 1∶1 ratio. Bright field and fluorescent microscopy photographs were taken on days 1–4 after addition of the MDA-MB-231 cells. Data are representative of experiments using three different ASC donors.

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: The effect of ASCs on the growth of MDA-MB 231 cells. A. MDA-MB-231 were cultured in the bottom well of a Boyden Chamber and ASCs were cultured in the insert. Growth of MDA-MB-231 cells was assessed using the MTT assay. B. 2.5×10 4 ASCs were cultured in 6 well plates for 24 hrs. prior to addition of MDA-MB-231-GFP breast cancer cells at a 1∶1 ratio. Bright field and fluorescent microscopy photographs were taken on days 1–4 after addition of the MDA-MB-231 cells. Data are representative of experiments using three different ASC donors.

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Multiple Displacement Amplification, Cell Culture, MTT Assay, Microscopy

    Metastatic lesions in lung and liver from the MDA-MB-231/GFP+ASC/RFP group tumors. 40 days after subcutaneous injection of MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected and 10 µM frozen sections were prepared for immunofluorescence of the lung and liver. A representative section of lung demonstrating multifocal metastatic lesions expressing GFP. A representative section of the liver demonstrated a small region expressing GFP. RFP was not detected above background level in any frozen tissue sections.

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: Metastatic lesions in lung and liver from the MDA-MB-231/GFP+ASC/RFP group tumors. 40 days after subcutaneous injection of MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected and 10 µM frozen sections were prepared for immunofluorescence of the lung and liver. A representative section of lung demonstrating multifocal metastatic lesions expressing GFP. A representative section of the liver demonstrated a small region expressing GFP. RFP was not detected above background level in any frozen tissue sections.

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Multiple Displacement Amplification, Injection, Immunofluorescence, Expressing

    ASC effect on primary MDA-MB-231 xenografts. 3×10 6 human MDA-MB-231/GFP breast cancer cells were bilaterally injected subcutaneously into the mammary fat pads of 5 female NUDE mice (n = 10 tumors/group) with or without 3×10 6 human ASC/RFP cells from donor with BMI 25.0 (A) or donor with BMI 18.3 (B).Tumor volume was monitored for 40 days by caliper measurement. Tumors were removed at day 40 and fluorescence of the intact, fresh tumors from the MDA-MB-231/GFP alone group ( C ) or MDA-MB-231/GFP+ASC/RFP group ( D ) were visualized for GFP and RFP within 10 minutes of removal using a dissecting fluorescent microscope. The white arrow indicates a region of RFP fluorescence only in the MDA-MB-231/GFP+ASC/RFP group tumors. E. 5 µM paraffin embedded section of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors were prepared for Hematoxylin and Eosin (H E) staining. F. 10 µM frozen sections of tumors were stained with DAPI (blue) and prepared for fluorescence microscopy for GFP and RFP. DAPI+GFP (DG); DAPI+RFP (DR); DAPI+GFP+RFP (DGR).

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: ASC effect on primary MDA-MB-231 xenografts. 3×10 6 human MDA-MB-231/GFP breast cancer cells were bilaterally injected subcutaneously into the mammary fat pads of 5 female NUDE mice (n = 10 tumors/group) with or without 3×10 6 human ASC/RFP cells from donor with BMI 25.0 (A) or donor with BMI 18.3 (B).Tumor volume was monitored for 40 days by caliper measurement. Tumors were removed at day 40 and fluorescence of the intact, fresh tumors from the MDA-MB-231/GFP alone group ( C ) or MDA-MB-231/GFP+ASC/RFP group ( D ) were visualized for GFP and RFP within 10 minutes of removal using a dissecting fluorescent microscope. The white arrow indicates a region of RFP fluorescence only in the MDA-MB-231/GFP+ASC/RFP group tumors. E. 5 µM paraffin embedded section of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors were prepared for Hematoxylin and Eosin (H E) staining. F. 10 µM frozen sections of tumors were stained with DAPI (blue) and prepared for fluorescence microscopy for GFP and RFP. DAPI+GFP (DG); DAPI+RFP (DR); DAPI+GFP+RFP (DGR).

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Multiple Displacement Amplification, Injection, Mouse Assay, Fluorescence, Microscopy, Staining

    ASC effect on migration of MDA-MB-231 cells. A. ASCs were cultured in the bottom well of a Boyden Chamber and MDA-MB-231 cells were cultured in the insert. Migration of MDA-MB-231 cells was assessed by using crystal violet staining of the insert membrane and quantification of color development. †P

    Journal: PLoS ONE

    Article Title: Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    doi: 10.1371/journal.pone.0089595

    Figure Lengend Snippet: ASC effect on migration of MDA-MB-231 cells. A. ASCs were cultured in the bottom well of a Boyden Chamber and MDA-MB-231 cells were cultured in the insert. Migration of MDA-MB-231 cells was assessed by using crystal violet staining of the insert membrane and quantification of color development. †P

    Article Snippet: Culture of MDA-MB-231 and MDA-MB-231/GFP cells MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and MDA-MB-231/GFP cells were purchased from Cell Biolabs, Inc. (San Diego, CA).

    Techniques: Migration, Multiple Displacement Amplification, Cell Culture, Staining

    Effect of alcohol on tumor angiogenesis in vitro . (A) The 3D tumor/endothelial cell co-culture system was set up as described in Materials and methods. Endothelial cells (SVECs or HUVECs) attached to cytodex beads were suspended in the fibrin gel containing SU5416 (0 or 2 μM) and breast cancer cells (E0771 or MDA-MB231) were placed on top of the gel. The endothelial cell sprouting was examined and recoded under a microscope following 12 h in culture. The percentage of beads with endothelial sprouts was calculated. EC, SVECs or HUVECs; TC, E0771 or MDA-MB231 cells. (B) SVEC/E0771 cell co-culture with/without SU5416 (2 μM) was exposed to alcohol (0 or 0.2%) for 12 h. Following this, the percentage of beads with endothelial sprouts was quantified. The experiment was replicated three times. (C) E0771 cells were maintained in a medium containing 1% FBS and exposed to alcohol (0 or 0.2%) for 24 h. The CM was collected and SVECs were incubated with this CM for 12 h. Then, the percentage of beads with endothelial sprouts was calculated. (D) The CM was collected from alcohol (0 or 0.2%)-treated E0771 cells and SU5416 (0 or 2 μM) was added to the medium. SVECs were then incubated with this conditioned medium for 12 h. The experiments were replicated three times. Each data point represents the mean ± SEM of three replicates. * P

    Journal: Oncology Letters

    Article Title: Alcohol promotes mammary tumor growth through activation of VEGF-dependent tumor angiogenesis

    doi: 10.3892/ol.2014.2146

    Figure Lengend Snippet: Effect of alcohol on tumor angiogenesis in vitro . (A) The 3D tumor/endothelial cell co-culture system was set up as described in Materials and methods. Endothelial cells (SVECs or HUVECs) attached to cytodex beads were suspended in the fibrin gel containing SU5416 (0 or 2 μM) and breast cancer cells (E0771 or MDA-MB231) were placed on top of the gel. The endothelial cell sprouting was examined and recoded under a microscope following 12 h in culture. The percentage of beads with endothelial sprouts was calculated. EC, SVECs or HUVECs; TC, E0771 or MDA-MB231 cells. (B) SVEC/E0771 cell co-culture with/without SU5416 (2 μM) was exposed to alcohol (0 or 0.2%) for 12 h. Following this, the percentage of beads with endothelial sprouts was quantified. The experiment was replicated three times. (C) E0771 cells were maintained in a medium containing 1% FBS and exposed to alcohol (0 or 0.2%) for 24 h. The CM was collected and SVECs were incubated with this CM for 12 h. Then, the percentage of beads with endothelial sprouts was calculated. (D) The CM was collected from alcohol (0 or 0.2%)-treated E0771 cells and SU5416 (0 or 2 μM) was added to the medium. SVECs were then incubated with this conditioned medium for 12 h. The experiments were replicated three times. Each data point represents the mean ± SEM of three replicates. * P

    Article Snippet: MDA-MB231 breast cancer cells and SVEC4-10EE2 murine endothelial cells (both American Type Culture Collection, Manassas, VA, USA) were grown in DMEM medium containing 10% FBS and 100 U/ml penicillin and streptomycin at 37°C with 5% CO2 .

    Techniques: In Vitro, Co-Culture Assay, Multiple Displacement Amplification, Microscopy, Incubation

    The effect of CVMSCs on MDA-MB231 cell migration using Boyden chamber migration assay. Where the control is C (MDA-MB231), and V is human placental chorionic villi derived MSC. The reduction in the migration response induced by CVMSCs was statistically significant (P = 0.037). Each experiment was repeated 3 times.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: The effect of CVMSCs on MDA-MB231 cell migration using Boyden chamber migration assay. Where the control is C (MDA-MB231), and V is human placental chorionic villi derived MSC. The reduction in the migration response induced by CVMSCs was statistically significant (P = 0.037). Each experiment was repeated 3 times.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Multiple Displacement Amplification, Migration, Derivative Assay

    Expression of cytokines and chemokines in pre-treated CVMSCs with MDA-MB231 through a transwell membrane. Two-way ANOVA analyzed the two treated groups; 1:1 and 1:3 and each group was compared to the control. The control is CVMSCs without any treatment. C is MDA-MB231, V is CVMSCs. CVMSCs = human placental chorionic villi derived MSCs.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: Expression of cytokines and chemokines in pre-treated CVMSCs with MDA-MB231 through a transwell membrane. Two-way ANOVA analyzed the two treated groups; 1:1 and 1:3 and each group was compared to the control. The control is CVMSCs without any treatment. C is MDA-MB231, V is CVMSCs. CVMSCs = human placental chorionic villi derived MSCs.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Expressing, Multiple Displacement Amplification, Derivative Assay

    Statistical computation of angiogenic parameters using angiogenesis analyzer macro connected image J software. The bars show a comparison between untreated MDA-MB231 and treated MDA-MB231 with different ration of CVMSCs in the presence of HUVECs or not. Nb = number. All the groups were analyzed by Two-way ANOVA and each group was compared to the positive group (HUVECs). HUVEC = human vascular endothelial cells, CVMSC = of human placental chorionic villi derived MSCs.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: Statistical computation of angiogenic parameters using angiogenesis analyzer macro connected image J software. The bars show a comparison between untreated MDA-MB231 and treated MDA-MB231 with different ration of CVMSCs in the presence of HUVECs or not. Nb = number. All the groups were analyzed by Two-way ANOVA and each group was compared to the positive group (HUVECs). HUVEC = human vascular endothelial cells, CVMSC = of human placental chorionic villi derived MSCs.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Software, Multiple Displacement Amplification, Derivative Assay

    Tube formation of CVMSCs and MDA-MB231 co-culture at different ratios. (A) Co-culture 1 CVMSCs and 1 MDA-MB231; (B) Co-culture 1 CVMSCs and 2 MDA-MB231 and; (C) Co-culture 3 CVMSCs and 1 MDA-MB231 on Matrigel with VEGF for 24h incubation. Images show CD31 (red) and Calcein AM staining (green) staining. 10x magnification for Calcein AM and 2x magnification for CD31.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: Tube formation of CVMSCs and MDA-MB231 co-culture at different ratios. (A) Co-culture 1 CVMSCs and 1 MDA-MB231; (B) Co-culture 1 CVMSCs and 2 MDA-MB231 and; (C) Co-culture 3 CVMSCs and 1 MDA-MB231 on Matrigel with VEGF for 24h incubation. Images show CD31 (red) and Calcein AM staining (green) staining. 10x magnification for Calcein AM and 2x magnification for CD31.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Multiple Displacement Amplification, Co-Culture Assay, Incubation, Staining

    CD31 and Calcein AM staining of individual cell population. (A) HUVECs; (B) MDA-MB231 and; (C) CVMSCs. Images show both CD31 (red) and Calcein AM staining (green) for HUVECs and MDA-MB231. Whereas CVMSCs, show no expression of CD31. (2x magnification for CD31 and 10x magnification for Calcein AM). 2x magnification for (C) .

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: CD31 and Calcein AM staining of individual cell population. (A) HUVECs; (B) MDA-MB231 and; (C) CVMSCs. Images show both CD31 (red) and Calcein AM staining (green) for HUVECs and MDA-MB231. Whereas CVMSCs, show no expression of CD31. (2x magnification for CD31 and 10x magnification for Calcein AM). 2x magnification for (C) .

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Staining, Multiple Displacement Amplification, Expressing

    A direct contact between CVMSCs and MDA-MB231 through a transwell membrane. CVMSCs were incubated overnight on the underside of the transwell. After 24 h the transwell was flipped over and MDA-MB231 cells were added on the upper chamber of the transwell and incubated for 48hr.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: A direct contact between CVMSCs and MDA-MB231 through a transwell membrane. CVMSCs were incubated overnight on the underside of the transwell. After 24 h the transwell was flipped over and MDA-MB231 cells were added on the upper chamber of the transwell and incubated for 48hr.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Multiple Displacement Amplification, Incubation

    Tube formation of CVMSCs, MDA-MB231 and HUVECs co-culture at different ratios. (A) Co-culture of 1 HUVECs: 1CVMSCs and 1 MDA-MB231; (B) Co-culture of 1 HUVECs: 1 CVMSCs and 2MDA-MB231 and; (C) Co-culture of 1 HUVECs: 2 CVMSCs and 1 MDA-MB231 and; (D) Co-culture of 1 HUVECs: 3 CVMSCs and 1 MDA-MB231 on Matrigel with VEGF for 24h incubation. Images show CD31 (red) and Calcein AM (green) staining or both overlay at 10x magnification.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: Tube formation of CVMSCs, MDA-MB231 and HUVECs co-culture at different ratios. (A) Co-culture of 1 HUVECs: 1CVMSCs and 1 MDA-MB231; (B) Co-culture of 1 HUVECs: 1 CVMSCs and 2MDA-MB231 and; (C) Co-culture of 1 HUVECs: 2 CVMSCs and 1 MDA-MB231 and; (D) Co-culture of 1 HUVECs: 3 CVMSCs and 1 MDA-MB231 on Matrigel with VEGF for 24h incubation. Images show CD31 (red) and Calcein AM (green) staining or both overlay at 10x magnification.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Multiple Displacement Amplification, Co-Culture Assay, Incubation, Staining

    Tube formation of individual cell population. (A) HUVECs; (B) MDA-MB231 and; (C) CVMSCs on Matrigel with VEGF for 24h incubation. Tubes started to form following 14 hour of incubation. 2x magnification.

    Journal: PLoS ONE

    Article Title: The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks

    doi: 10.1371/journal.pone.0207593

    Figure Lengend Snippet: Tube formation of individual cell population. (A) HUVECs; (B) MDA-MB231 and; (C) CVMSCs on Matrigel with VEGF for 24h incubation. Tubes started to form following 14 hour of incubation. 2x magnification.

    Article Snippet: The MDA-MB231 TNBC cell line was used for this study (ATCC, USA).

    Techniques: Multiple Displacement Amplification, Incubation

    Acetylation of H2A.Z at the p21 promoter is necessary for its transcription. a) Schematic representation of the p21 promoter region showing PCR amplified fragments (1–4). b) Binding of H2A.Z to the p21 promoter in MDA-MB231 cells. c) mRNA expression of H2AFZ (left) and p21 (right) in MDA-MB231 cells transfected with a smartpool siH2A.Z for 72h. d) H2A.Z and polymerase II (pol II) binding to the p21 TSS (fragment #4) in cells transfected with siH2A.Z or scramble siRNAs. e) H2A.Z binding to the p21 TSS (fragment #4) in cells treated with TSA (50 ng/ml) for 48 h. f–g) acetylated H2A.Z, polymerase II (pol II) (f) and acetylated H3K9 (g) binding to the p21 TSS (fragment #4) in cells treated with TSA. h-i) acetylated H2A.Z (h) and H3K9 (i) amount at p21 TSS in cells transfected with siH2A.Z or scramble siRNA.

    Journal: PLoS ONE

    Article Title: Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z

    doi: 10.1371/journal.pone.0054102

    Figure Lengend Snippet: Acetylation of H2A.Z at the p21 promoter is necessary for its transcription. a) Schematic representation of the p21 promoter region showing PCR amplified fragments (1–4). b) Binding of H2A.Z to the p21 promoter in MDA-MB231 cells. c) mRNA expression of H2AFZ (left) and p21 (right) in MDA-MB231 cells transfected with a smartpool siH2A.Z for 72h. d) H2A.Z and polymerase II (pol II) binding to the p21 TSS (fragment #4) in cells transfected with siH2A.Z or scramble siRNAs. e) H2A.Z binding to the p21 TSS (fragment #4) in cells treated with TSA (50 ng/ml) for 48 h. f–g) acetylated H2A.Z, polymerase II (pol II) (f) and acetylated H3K9 (g) binding to the p21 TSS (fragment #4) in cells treated with TSA. h-i) acetylated H2A.Z (h) and H3K9 (i) amount at p21 TSS in cells transfected with siH2A.Z or scramble siRNA.

    Article Snippet: MDA-MB231 cells were treated with 50 or 100 ng/ml TSA (Sigma-Aldrich) and with LBH589 5×10−8 or 5×10−9 M for the indicated times.

    Techniques: Polymerase Chain Reaction, Amplification, Binding Assay, Multiple Displacement Amplification, Expressing, Transfection

    p400 but not Tip60 functions in p21 expression in the absence of p53. a) Tip60 and p21 mRNA expression. b) H2A.Z, Acetyl-H2A.Z and RNA pol II binding to the p21 TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the p21 TSS (fragment #4) in cells treated with TSA. d) p21 mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e) H2A.Z, Acetyl-H2A.Z and p300 enrichment at p21 TSS (fragment #4) in MDA-MB231 transfected with si p400.

    Journal: PLoS ONE

    Article Title: Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z

    doi: 10.1371/journal.pone.0054102

    Figure Lengend Snippet: p400 but not Tip60 functions in p21 expression in the absence of p53. a) Tip60 and p21 mRNA expression. b) H2A.Z, Acetyl-H2A.Z and RNA pol II binding to the p21 TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the p21 TSS (fragment #4) in cells treated with TSA. d) p21 mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e) H2A.Z, Acetyl-H2A.Z and p300 enrichment at p21 TSS (fragment #4) in MDA-MB231 transfected with si p400.

    Article Snippet: MDA-MB231 cells were treated with 50 or 100 ng/ml TSA (Sigma-Aldrich) and with LBH589 5×10−8 or 5×10−9 M for the indicated times.

    Techniques: Expressing, Binding Assay, Transfection, Chromatin Immunoprecipitation, Multiple Displacement Amplification

    HDAC inhibitors reduce proliferation and activate p21 transcription in ERα- negative/p53 mutated mammary tumor cells. a) MTT assay to quantify proliferation rates in the presence of Trichostatin A of MDA-MB231 cells. Two different concentrations (50 ng/ml and 100 ng/ml) were used. b-c) q-PCR and western blot analysis of p21 mRNA expression and protein levels. Experiments were performed three times.

    Journal: PLoS ONE

    Article Title: Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z

    doi: 10.1371/journal.pone.0054102

    Figure Lengend Snippet: HDAC inhibitors reduce proliferation and activate p21 transcription in ERα- negative/p53 mutated mammary tumor cells. a) MTT assay to quantify proliferation rates in the presence of Trichostatin A of MDA-MB231 cells. Two different concentrations (50 ng/ml and 100 ng/ml) were used. b-c) q-PCR and western blot analysis of p21 mRNA expression and protein levels. Experiments were performed three times.

    Article Snippet: MDA-MB231 cells were treated with 50 or 100 ng/ml TSA (Sigma-Aldrich) and with LBH589 5×10−8 or 5×10−9 M for the indicated times.

    Techniques: MTT Assay, Multiple Displacement Amplification, Polymerase Chain Reaction, Western Blot, Expressing

    MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P

    Journal: American Journal of Cancer Research

    Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

    doi:

    Figure Lengend Snippet: MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P

    Article Snippet: MDA-MB231 and SKBR3 cells were grown to 70% confluence before being transfected with viral supernatants containing 5 mg/ml polybrene (Sigma, MO).

    Techniques: Multiple Displacement Amplification, shRNA, Transfection, Quantitation Assay

    CBD modulates expression of prohibitin and STAT3 in cancer cells. PC3, HEPG2, and MDA-MB-231 cells were tested for changes in mitochondrial associated prohibitin and STAT3 expression following 1 h treatment with CBD (5 μM). Levels of prohibitin were reduced in all three cancer cell lines while this reduction was more marked in PC3 (A) and HEPG2 (B) cells compared to MDA-MB-231 (C) . STAT3 (phospho Y705) was also reduced after 1 h CBD (5 μM) in both PC3 (D) and HEPG2 cells (E) , while MDA-MB-231 cells showed a similar, albeit less marked trend (F) . Beta-actin is shown as an internal loading control and “R” indicates the change of prohibitin and pSTAT3 expression relative to β-actin levels, respectively, for comparison between CBD treatment and DMSO control.

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CBD modulates expression of prohibitin and STAT3 in cancer cells. PC3, HEPG2, and MDA-MB-231 cells were tested for changes in mitochondrial associated prohibitin and STAT3 expression following 1 h treatment with CBD (5 μM). Levels of prohibitin were reduced in all three cancer cell lines while this reduction was more marked in PC3 (A) and HEPG2 (B) cells compared to MDA-MB-231 (C) . STAT3 (phospho Y705) was also reduced after 1 h CBD (5 μM) in both PC3 (D) and HEPG2 cells (E) , while MDA-MB-231 cells showed a similar, albeit less marked trend (F) . Beta-actin is shown as an internal loading control and “R” indicates the change of prohibitin and pSTAT3 expression relative to β-actin levels, respectively, for comparison between CBD treatment and DMSO control.

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Expressing, Multiple Displacement Amplification

    CBD sensitizes HEPG2 and MD-MB-231 cancer cells to cisplatin-mediated apoptosis. HEPG2 (A) and MDA-MB-231 (B) cells were treated with 1 or 5 μM CBD for 24 h prior to further 24 h incubation with cisplatin (CSP, 100 μM). Cell viability was assessed by MTT assay. Data shown is repeated three times with three technical replicates per plate. Data is represented as mean ± SEM. ∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CBD sensitizes HEPG2 and MD-MB-231 cancer cells to cisplatin-mediated apoptosis. HEPG2 (A) and MDA-MB-231 (B) cells were treated with 1 or 5 μM CBD for 24 h prior to further 24 h incubation with cisplatin (CSP, 100 μM). Cell viability was assessed by MTT assay. Data shown is repeated three times with three technical replicates per plate. Data is represented as mean ± SEM. ∗ p

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Multiple Displacement Amplification, Incubation, MTT Assay

    CBD modulates different MV subpopulations released from PC3, HEPG2 and MDA-MB-231 cells. Inhibitory effects of CBD alone, Cl-amidine or CBD in combination with Cl-amidine on 100–200 nm and 201–500 nm sized microvesicles, based on size exclusion analysis by Nanosight Tracking Analysis (NTA). Inhibition of 100–200 nm MV release in (A) PC3; (C) HEPG2; and (E) MDA-MB-231 cancer cells. Inhibition of 201–500 nm MV release in (B) PC3; (D) HEPG2; and (F) MDA-MB-231 cancer cells. The experiments were repeated three times and the data presented are mean ± SEM of the results ( ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001 versus Control; Differences between CBD and Cl-amidine treatment group is further indicated as # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001; #### p ≤ 0.0001).

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CBD modulates different MV subpopulations released from PC3, HEPG2 and MDA-MB-231 cells. Inhibitory effects of CBD alone, Cl-amidine or CBD in combination with Cl-amidine on 100–200 nm and 201–500 nm sized microvesicles, based on size exclusion analysis by Nanosight Tracking Analysis (NTA). Inhibition of 100–200 nm MV release in (A) PC3; (C) HEPG2; and (E) MDA-MB-231 cancer cells. Inhibition of 201–500 nm MV release in (B) PC3; (D) HEPG2; and (F) MDA-MB-231 cancer cells. The experiments were repeated three times and the data presented are mean ± SEM of the results ( ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001 versus Control; Differences between CBD and Cl-amidine treatment group is further indicated as # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001; #### p ≤ 0.0001).

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Multiple Displacement Amplification, Inhibition

    CBD significantly inhibits total EMV, exosome and MV release from MDA-MB-231 cells. Inhibitory effects of CBD alone and in combination with Cl-amidine on extracellular vesicle release from MDA-MB-231 cancer cells are presented as histograms which are based on size exclusion analysis by Nanosight Tracking Analysis (NTA). EMVs represent all vesicles 0–900 nm (A) ; exosomes are vesicles

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CBD significantly inhibits total EMV, exosome and MV release from MDA-MB-231 cells. Inhibitory effects of CBD alone and in combination with Cl-amidine on extracellular vesicle release from MDA-MB-231 cancer cells are presented as histograms which are based on size exclusion analysis by Nanosight Tracking Analysis (NTA). EMVs represent all vesicles 0–900 nm (A) ; exosomes are vesicles

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Multiple Displacement Amplification

    CD63 exosomal marker is reduced following 1 h CBD treatment in HEPG2, PC3, and MDA-MB-231 cancer cells. The results from the NTA analysis were confirmed by Western blotting for the exosomal CD63 marker, which was reduced in all three cell lines following 1 h treatment with 5 μM CBD: (A) CD63 expression is reduced in CBD treated versus DMSO control treated HEPG2 cancer cells; (B) CD63 expression is reduced in CBD treated versus DMSO control treated PC3 cells; (C) CD63 expression is reduced in CBD treated versus DMSO control treated MDA-MB-231 cells. All EMV preparations were performed in equal buffer volume (50 μl) and all cell lysates were prepared in equal buffer volume (50 μl) between all samples, for accurate presentation of amounts of vesicles isolated and amounts of cells grown and collected per flask. For EMV isolates, 20 μl of sample was loaded per lane, while for β-actin detection in the corresponding cell lysates, 10 μl of sample was loaded per lane. The relative detection of CD63 in EMVs released from the corresponding cell preparation is indicated by “R,” in relation to β-actin detection in the corresponding cell isolate, for comparison between CBD treatments versus DMSO control.

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CD63 exosomal marker is reduced following 1 h CBD treatment in HEPG2, PC3, and MDA-MB-231 cancer cells. The results from the NTA analysis were confirmed by Western blotting for the exosomal CD63 marker, which was reduced in all three cell lines following 1 h treatment with 5 μM CBD: (A) CD63 expression is reduced in CBD treated versus DMSO control treated HEPG2 cancer cells; (B) CD63 expression is reduced in CBD treated versus DMSO control treated PC3 cells; (C) CD63 expression is reduced in CBD treated versus DMSO control treated MDA-MB-231 cells. All EMV preparations were performed in equal buffer volume (50 μl) and all cell lysates were prepared in equal buffer volume (50 μl) between all samples, for accurate presentation of amounts of vesicles isolated and amounts of cells grown and collected per flask. For EMV isolates, 20 μl of sample was loaded per lane, while for β-actin detection in the corresponding cell lysates, 10 μl of sample was loaded per lane. The relative detection of CD63 in EMVs released from the corresponding cell preparation is indicated by “R,” in relation to β-actin detection in the corresponding cell isolate, for comparison between CBD treatments versus DMSO control.

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Marker, Multiple Displacement Amplification, Western Blot, Expressing, Isolation

    CBD does not affect cell viability of PC3, HEPG2, and MDA-MB-231 cells after 1 h treatment. The Guava EasyCyte 8HT flow cytometer (Millipore) and ViaCount assay were used to count and determine viability of CBD treated cells compared to EMV inhibitor Cl-amidine and DMSO treated control cells after 1 h incubation ( ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001).

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: CBD does not affect cell viability of PC3, HEPG2, and MDA-MB-231 cells after 1 h treatment. The Guava EasyCyte 8HT flow cytometer (Millipore) and ViaCount assay were used to count and determine viability of CBD treated cells compared to EMV inhibitor Cl-amidine and DMSO treated control cells after 1 h incubation ( ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001).

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Incubation

    Mitochondrial function alteration following 1 h CBD treatment in MDA-MB-231 and PC3 cancer cells. MDA-MB-231 and PC3 cells were treated with 1 or 5 μM CBD for 1 h prior to mitochondrial functional analysis using the Seahorse Bioanalyser for the following parameters: (A,D) Basal mitochondrial respiration; (B,E) Quantification of ATP production; (C,F) Proton leak. Data shown is repeated three times (for MDA-MB-231 cells) or five times (for PC3 cells) with four technical replicates per plate. Data is represented as mean ± SEM. ∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer

    doi: 10.3389/fphar.2018.00889

    Figure Lengend Snippet: Mitochondrial function alteration following 1 h CBD treatment in MDA-MB-231 and PC3 cancer cells. MDA-MB-231 and PC3 cells were treated with 1 or 5 μM CBD for 1 h prior to mitochondrial functional analysis using the Seahorse Bioanalyser for the following parameters: (A,D) Basal mitochondrial respiration; (B,E) Quantification of ATP production; (C,F) Proton leak. Data shown is repeated three times (for MDA-MB-231 cells) or five times (for PC3 cells) with four technical replicates per plate. Data is represented as mean ± SEM. ∗ p

    Article Snippet: Effects on EMV Biogenesis Using CBD and Cl-Amidine For assessment of effects of CBD and Cl-amidine on EMV generation, PC3, HEPG2 and MDA-MB-231 cells were seeded at a density of 3.8 × 105 cells/well, in triplicate, in 12-well microtiter plates, using pre-warmed serum- and EMV-free RPMI 1640 (Sigma-Aldrich, United Kingdom).

    Techniques: Multiple Displacement Amplification, Functional Assay

    Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation ( A,B ). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX ( C ) RING1B ( D ) and Ub-H2A ( E ) p53BP ( F ) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation ( A,B ). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX ( C ) RING1B ( D ) and Ub-H2A ( E ) p53BP ( F ) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Western Blot, Expressing, Transfection, Immunofluorescence

    Ectopic expression of miR-15a/16 intensifies DNA damage and repair. Comet assay in MCF-7 and MDAMB-231 cells after transfection with miR-15a, miR-16, both miR-15a/16 and anti-miR-15a, anti-miR-16 and both. Etoposide was used to chemically induced DNA damage. Cells were processed for single-cell gel electrophoresis (comet assay) and miR-15a, miR-16 or both miR-15a/16 transfected cells show an increase in DNA damage as compared to cells transfected with scrambled miRNAs and anti-miRs under same damage condition. Bar indicates 150 µm ( A,B ). Cells with DNA damage that showed prominent tail (Comet) were scored using cometIV software and percentage of tail DNA was plotted ( C,D,E,F ).

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: Ectopic expression of miR-15a/16 intensifies DNA damage and repair. Comet assay in MCF-7 and MDAMB-231 cells after transfection with miR-15a, miR-16, both miR-15a/16 and anti-miR-15a, anti-miR-16 and both. Etoposide was used to chemically induced DNA damage. Cells were processed for single-cell gel electrophoresis (comet assay) and miR-15a, miR-16 or both miR-15a/16 transfected cells show an increase in DNA damage as compared to cells transfected with scrambled miRNAs and anti-miRs under same damage condition. Bar indicates 150 µm ( A,B ). Cells with DNA damage that showed prominent tail (Comet) were scored using cometIV software and percentage of tail DNA was plotted ( C,D,E,F ).

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Single Cell Gel Electrophoresis, Transfection, Software

    Rescue of BMI1 level as well as inhibiting endogenous miR-15a, miR-16 reduces miR-15a, miR-16 mediated DNA damage. Expression of BMI1, ϒ-H2AX, p-ATM, Ub-H2A and RNF8 were checked in MCF-7, MDAMB-231 cells transfected with anti-miR-15a, anti-miR-16 or both anti-miR-15a/16. Actin served as gel loading control. Blots were cropped to enhance the representation ( A ). Expression of BMI1, ϒ-H2AX, Ub-H2A, RNF8, p-CHK2, p-ATM were checked in MDAMB-231 cells co-transfected with miR-15a, miR-16, miR-15a/16 and pT3-EF1a-Bmi1 plasmid that lack 3′UTR. Scramble miRNA vector used to serve as a control. Actin is used as a gel loading control. Blots were cropped to enhance the representation ( B ). Immunofluorescence studies showing accumulation of BMI1, ϒ-H2AX ( C ), Ub-H2A ( D ) in MDAMB-231 cells transfected with anti-miR-15a anti-miR-16 or both anti-miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm. Expression of BMI1, Ub-H2A, γ-H2AX, p-CHK2, RNF8 were checked in MDAMB-231 cells transfected with either scrambled si-RNA or si-RNA specifically against BMI1. Actin is used as a gel loading control. Blots were cropped to enhance the representation ( E ). Immunofluorescence studies showing the localization of BMI1 and γ-H2AX foci in cells treated with scramble si-RNA (si-scrambled) or si-RNA against BMI1 (si-BMI1). Cells show an increase in γ-H2AX foci upon depletion of BMI1. Bar indicates 200 μm ( F ).

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: Rescue of BMI1 level as well as inhibiting endogenous miR-15a, miR-16 reduces miR-15a, miR-16 mediated DNA damage. Expression of BMI1, ϒ-H2AX, p-ATM, Ub-H2A and RNF8 were checked in MCF-7, MDAMB-231 cells transfected with anti-miR-15a, anti-miR-16 or both anti-miR-15a/16. Actin served as gel loading control. Blots were cropped to enhance the representation ( A ). Expression of BMI1, ϒ-H2AX, Ub-H2A, RNF8, p-CHK2, p-ATM were checked in MDAMB-231 cells co-transfected with miR-15a, miR-16, miR-15a/16 and pT3-EF1a-Bmi1 plasmid that lack 3′UTR. Scramble miRNA vector used to serve as a control. Actin is used as a gel loading control. Blots were cropped to enhance the representation ( B ). Immunofluorescence studies showing accumulation of BMI1, ϒ-H2AX ( C ), Ub-H2A ( D ) in MDAMB-231 cells transfected with anti-miR-15a anti-miR-16 or both anti-miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm. Expression of BMI1, Ub-H2A, γ-H2AX, p-CHK2, RNF8 were checked in MDAMB-231 cells transfected with either scrambled si-RNA or si-RNA specifically against BMI1. Actin is used as a gel loading control. Blots were cropped to enhance the representation ( E ). Immunofluorescence studies showing the localization of BMI1 and γ-H2AX foci in cells treated with scramble si-RNA (si-scrambled) or si-RNA against BMI1 (si-BMI1). Cells show an increase in γ-H2AX foci upon depletion of BMI1. Bar indicates 200 μm ( F ).

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Transfection, Plasmid Preparation, Immunofluorescence

    miR-15a/16 sensitizes the breast cancer cells to the chemotherapeutic drug Protein expression of BMI1, Ub-H2A, BID, BCL2, Caspase-3, p-AKT, GSK3β were checked in MDAMB-231 cells ectopically expressed with miR-15a, miR-16 or both miR-15a/16 and treated with doxorubicin. Actin served as gel loading control. Scramble miRNA vector was used as transfection control. Blots were cropped to enhance the representation ( A ). Results of MTT assay showing the effect of miR-15a, miR-16 treated with doxorubicin in MDAMB-231 cells. Scramble miRNA vector was used as control. Error bars indicate ± S.E. of n = 3 ( B ). Tunel assay showing the apoptotic cascade of miR-15a, miR-16 ectopically expressed in MDAMB-231 cells treated with doxorubicin. Scramble miRNA vector was used as control. Bar indicates 200 μm ( C ). Acridine orange/Ethidium bromide double staining represents the live (Green), early apoptotic (Yellow) and late apoptotic/dead (orange/red) cells of miR-15a, miR-16 ectopically expressed in MDAMB-231 cells treated with doxorubicin. Bar indicates 100 μm ( D ).

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: miR-15a/16 sensitizes the breast cancer cells to the chemotherapeutic drug Protein expression of BMI1, Ub-H2A, BID, BCL2, Caspase-3, p-AKT, GSK3β were checked in MDAMB-231 cells ectopically expressed with miR-15a, miR-16 or both miR-15a/16 and treated with doxorubicin. Actin served as gel loading control. Scramble miRNA vector was used as transfection control. Blots were cropped to enhance the representation ( A ). Results of MTT assay showing the effect of miR-15a, miR-16 treated with doxorubicin in MDAMB-231 cells. Scramble miRNA vector was used as control. Error bars indicate ± S.E. of n = 3 ( B ). Tunel assay showing the apoptotic cascade of miR-15a, miR-16 ectopically expressed in MDAMB-231 cells treated with doxorubicin. Scramble miRNA vector was used as control. Bar indicates 200 μm ( C ). Acridine orange/Ethidium bromide double staining represents the live (Green), early apoptotic (Yellow) and late apoptotic/dead (orange/red) cells of miR-15a, miR-16 ectopically expressed in MDAMB-231 cells treated with doxorubicin. Bar indicates 100 μm ( D ).

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Plasmid Preparation, Transfection, MTT Assay, TUNEL Assay, Double Staining

    Ectopic expression of miR-15a, miR-16 sensitizes breast cancer cells to DNA damage through BMI1 dependent ubiquitination pathway. Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231 cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation ( A,B ). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX ( C ) RING1B ( D ) and Ub-H2A ( E ) p53BP ( F ) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: Ectopic expression of miR-15a, miR-16 sensitizes breast cancer cells to DNA damage through BMI1 dependent ubiquitination pathway. Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231 cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation ( A,B ). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX ( C ) RING1B ( D ) and Ub-H2A ( E ) p53BP ( F ) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Western Blot, Transfection, Immunofluorescence

    Time-dependent expression of proteins after ectopic expression of miR-15a/16 and overexpression of miR-15a/16 impedes homologous recombination-mediated repair. Western blot analysis showing the expression of BMI1, γ-H2AX, Ub-H2A in MDAMB-231 cells transfected with both miR-15a/16 followed by treatment with etoposide for 2 to 72 hrs. Actin was used as a gel loading control. During the course of time periods (0 hrs to 72 hrs). Blots were cropped to enhance the representation ( A ). Heat map showing the endogenous expression of quantitative RT-PCR analysis. The Green colour in the heat map indicates decreased and red colour indicates increased mRNA expression level. Gene specific primers were used for the amplification. GAPDH was used as endogenous control ( B,C ) Metaphase chromosomes of cells overexpressing Mir-15a/16. (i) Metaphase fromcontrol, (ii) Metaphase from irradiated cell showing gaps (small arrow) and radials (large arrows)in human H1299 cells ( D ). In human H1299 cells S-phase specific chromosome aberrations after 2 Gy irradiations. * p

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: Time-dependent expression of proteins after ectopic expression of miR-15a/16 and overexpression of miR-15a/16 impedes homologous recombination-mediated repair. Western blot analysis showing the expression of BMI1, γ-H2AX, Ub-H2A in MDAMB-231 cells transfected with both miR-15a/16 followed by treatment with etoposide for 2 to 72 hrs. Actin was used as a gel loading control. During the course of time periods (0 hrs to 72 hrs). Blots were cropped to enhance the representation ( A ). Heat map showing the endogenous expression of quantitative RT-PCR analysis. The Green colour in the heat map indicates decreased and red colour indicates increased mRNA expression level. Gene specific primers were used for the amplification. GAPDH was used as endogenous control ( B,C ) Metaphase chromosomes of cells overexpressing Mir-15a/16. (i) Metaphase fromcontrol, (ii) Metaphase from irradiated cell showing gaps (small arrow) and radials (large arrows)in human H1299 cells ( D ). In human H1299 cells S-phase specific chromosome aberrations after 2 Gy irradiations. * p

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Over Expression, Homologous Recombination, Western Blot, Transfection, Quantitative RT-PCR, Amplification, Irradiation

    miR-15a/16 expression is altered in chemically induced DNA damage and it suppresses the BMI1 expression by targeting 3′UTR. Heat map showing the endogenous expression of miRNAs. The Green colour in the heat map indicates decreased and red colour indicates increased mRNA expression level. U6 was used as endogenous control ( A ). Predicted miRNA sequences and their recognition sites within 3′UTR of BMI1 ( B ) Renilla luciferase reporter assay showing the reporter expression in MCF-7 and MDAMB-231 cells co-transfected with wild-type 3′UTR of BMI1and mutant 3′UTR of BMI1 along with miR-15a, miR-16. Scramble miRNA vector used as a control. Renilla luciferase activity was normalized to firefly luciferase. n = 3, Students t-test was used to generate P values, *, ** and *** indicates P value summary ( C,D ). Western blot and RT-PCR data showing the expression of BMI1 upon overexpressed miR-15a, miR-16 and both. Blots were cropped to enhance the representation ( E ).

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: miR-15a/16 expression is altered in chemically induced DNA damage and it suppresses the BMI1 expression by targeting 3′UTR. Heat map showing the endogenous expression of miRNAs. The Green colour in the heat map indicates decreased and red colour indicates increased mRNA expression level. U6 was used as endogenous control ( A ). Predicted miRNA sequences and their recognition sites within 3′UTR of BMI1 ( B ) Renilla luciferase reporter assay showing the reporter expression in MCF-7 and MDAMB-231 cells co-transfected with wild-type 3′UTR of BMI1and mutant 3′UTR of BMI1 along with miR-15a, miR-16. Scramble miRNA vector used as a control. Renilla luciferase activity was normalized to firefly luciferase. n = 3, Students t-test was used to generate P values, *, ** and *** indicates P value summary ( C,D ). Western blot and RT-PCR data showing the expression of BMI1 upon overexpressed miR-15a, miR-16 and both. Blots were cropped to enhance the representation ( E ).

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Mutagenesis, Plasmid Preparation, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    miR-15a/16 mediated of G2/M checkpoints activation. miR-15a, miR-16 and both miR-15a/16 were overexpressed in MDAMB-231 cells and cell cycle analysis was performed. Scramble miRNA vector used as a control ( A ). Anti-miR-15a, anti-miR-16 and both anti-miR-15a/16 were overexpressed in MDAMB-231 cells and cell cycle analysis was performed. Scramble miRNA vector used as a control ( B ). Graph bars represent the percentage of cells in each cell cycle phase ( C,D ). In overexpressed miR-15a, miR-16 and miR-15a/16 cells p21, p53, p53 (S46), p53 (S15), CDK1, Cyclin-B1 protein expression levels were checked. Actin served as gel loading control. Blots were cropped to enhance the representation. Scramble miRNA vector was used as transfection control ( E ).

    Journal: Scientific Reports

    Article Title: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

    doi: 10.1038/s41598-017-02800-2

    Figure Lengend Snippet: miR-15a/16 mediated of G2/M checkpoints activation. miR-15a, miR-16 and both miR-15a/16 were overexpressed in MDAMB-231 cells and cell cycle analysis was performed. Scramble miRNA vector used as a control ( A ). Anti-miR-15a, anti-miR-16 and both anti-miR-15a/16 were overexpressed in MDAMB-231 cells and cell cycle analysis was performed. Scramble miRNA vector used as a control ( B ). Graph bars represent the percentage of cells in each cell cycle phase ( C,D ). In overexpressed miR-15a, miR-16 and miR-15a/16 cells p21, p53, p53 (S46), p53 (S15), CDK1, Cyclin-B1 protein expression levels were checked. Actin served as gel loading control. Blots were cropped to enhance the representation. Scramble miRNA vector was used as transfection control ( E ).

    Article Snippet: MCF-7 cells were maintained in DMEM and MDAMB-231 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) 100 U/ml, Penicillin and 100 mg/ml streptomycin sulfate (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere.

    Techniques: Activation Assay, Cell Cycle Assay, Plasmid Preparation, Expressing, Transfection

    The HJURP mRNA level in breast cancer cell lines predicts the sensitivity to radiation treatment . Part (a) shows the percent of viable cells at 72 hours after different doses of radiation in breast cancer cell line MDAMB231 with a high level of HJURP and T47D with a low level of HJURP . MDAMD231 cells are more sensitive to radiation treatment than T47D cells. Figure 7 b shows the fold change of apoptosis in comparison to control (no radiation) at 72 hours after the different dose of radiation in breast cancer cell line MDAMB231 and T47D. There are more apoptosis in MDAMB231 cells than T47D cells. Part (c) shows the percent of viable cells at 72 hours after the different dose of radiation in breast cancer cell line BT20 with high level of HJURP and MCF10A with low level of HJURP . BT20 cells are more sensitive to radiation treatment than MCF10A cells. Part (d) shows the fold change of apoptosis in comparison to control (no radiation) at 72 hours after the different dose of radiation in breast cancer cell line BT20 and MCF10A. There are more apoptosis in BT20 cells than MCF10A cells. (e) HJURP protein levels are down-regulated by shRNA in MDAMB231 breast cancer cell lines. Part (f) shows that MDAMB231 breast cancer cells with shRNA against HJURP reduce the sensitivity to radiation.

    Journal: Breast Cancer Research : BCR

    Article Title: The expression level of HJURP has an independent prognostic impact and predicts the sensitivity to radiotherapy in breast cancer

    doi: 10.1186/bcr2487

    Figure Lengend Snippet: The HJURP mRNA level in breast cancer cell lines predicts the sensitivity to radiation treatment . Part (a) shows the percent of viable cells at 72 hours after different doses of radiation in breast cancer cell line MDAMB231 with a high level of HJURP and T47D with a low level of HJURP . MDAMD231 cells are more sensitive to radiation treatment than T47D cells. Figure 7 b shows the fold change of apoptosis in comparison to control (no radiation) at 72 hours after the different dose of radiation in breast cancer cell line MDAMB231 and T47D. There are more apoptosis in MDAMB231 cells than T47D cells. Part (c) shows the percent of viable cells at 72 hours after the different dose of radiation in breast cancer cell line BT20 with high level of HJURP and MCF10A with low level of HJURP . BT20 cells are more sensitive to radiation treatment than MCF10A cells. Part (d) shows the fold change of apoptosis in comparison to control (no radiation) at 72 hours after the different dose of radiation in breast cancer cell line BT20 and MCF10A. There are more apoptosis in BT20 cells than MCF10A cells. (e) HJURP protein levels are down-regulated by shRNA in MDAMB231 breast cancer cell lines. Part (f) shows that MDAMB231 breast cancer cells with shRNA against HJURP reduce the sensitivity to radiation.

    Article Snippet: Retroviral infection was performed by adding filtered supernatant to a MDAMB231 cell line cultured on 10 cm dishes with 50% confluent in the presence 4 ug/ml of polybrene (Sigma, St. Louis, MO, USA).

    Techniques: shRNA

    Loss of REST leads to increased IRS1 protein levels. (A) Western blot images showing REST and IRS1 expression in MCF7 (ER + ), MDA-MB-231 (ER − ), and ZR751 (ER + ) cells. (B) Quantifications of relative protein expression depicted in a graph (for shRNA

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: Loss of REST leads to increased IRS1 protein levels. (A) Western blot images showing REST and IRS1 expression in MCF7 (ER + ), MDA-MB-231 (ER − ), and ZR751 (ER + ) cells. (B) Quantifications of relative protein expression depicted in a graph (for shRNA

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Western Blot, Expressing, Multiple Displacement Amplification, shRNA

    Induced REST expression represses IRS1 mRNA in MDA-MB-231 cells but not in MCF7 cells. (A) REST and IRS1 mRNAs extracted from MDA-MB-231 cells expressing Tet-inducible REST. Significance was determined by using a t test ( n = 3). (B) Western blotting of

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: Induced REST expression represses IRS1 mRNA in MDA-MB-231 cells but not in MCF7 cells. (A) REST and IRS1 mRNAs extracted from MDA-MB-231 cells expressing Tet-inducible REST. Significance was determined by using a t test ( n = 3). (B) Western blotting of

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot

    IRS1 activity is necessary for enhanced signaling and growth in MCF7 and MDA-MB-231 cells. (A) Schematic of protein domains in the IRS1-DN construct compared to full-length IRS1. The domains conserved in the IRS1-DN are the pleckstrin homology (PH) domain

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: IRS1 activity is necessary for enhanced signaling and growth in MCF7 and MDA-MB-231 cells. (A) Schematic of protein domains in the IRS1-DN construct compared to full-length IRS1. The domains conserved in the IRS1-DN are the pleckstrin homology (PH) domain

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Activity Assay, Multiple Displacement Amplification, Construct

    Cells lacking REST have enhanced signaling in response to IGF. Shown are Western blot images from lysates of REST norm and REST low cells treated with IGF for 10 min for MCF7 cells (A) or for 2 h for MDA-MB-231 cells (B) ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: Cells lacking REST have enhanced signaling in response to IGF. Shown are Western blot images from lysates of REST norm and REST low cells treated with IGF for 10 min for MCF7 cells (A) or for 2 h for MDA-MB-231 cells (B) ( n = 3).

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Western Blot, Multiple Displacement Amplification

    REST directly represses IRS1. (A) Primer sites flanking the RE-1 site positioned 12.4 kb upstream of the IRS1 promoter were used for ChIP and RE-1 reporter assays. (B and C) ChIP of REST at GAPDH, BDNF, and IRS1 in MCF7 (B) and MDA-MB-231 (C) cells (

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: REST directly represses IRS1. (A) Primer sites flanking the RE-1 site positioned 12.4 kb upstream of the IRS1 promoter were used for ChIP and RE-1 reporter assays. (B and C) ChIP of REST at GAPDH, BDNF, and IRS1 in MCF7 (B) and MDA-MB-231 (C) cells (

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Chromatin Immunoprecipitation, Multiple Displacement Amplification

    Heightened signaling of PI3K/AKT and MAPK/ERK is required for enhanced growth of REST low cells. (A) Inhibitors of the IGF1R/IRS1 pathway. (B and C) Soft-agar growth with inhibitor treatment in REST norm and REST low MCF7 ( n = 27) (B) or MDA-MB-231 ( n =

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: Heightened signaling of PI3K/AKT and MAPK/ERK is required for enhanced growth of REST low cells. (A) Inhibitors of the IGF1R/IRS1 pathway. (B and C) Soft-agar growth with inhibitor treatment in REST norm and REST low MCF7 ( n = 27) (B) or MDA-MB-231 ( n =

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Multiple Displacement Amplification

    Loss of REST confers cell-type-specific phenotypes. (A and B) Relative absorbance of crystal violet stain representing cell numbers for MCF7 ( n = 24) (A) and MDA-MB-231 ( n = 18) (B) cells, expressed as a signal proportional to the REST norm , untreated

    Journal: Molecular and Cellular Biology

    Article Title: Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

    doi: 10.1128/MCB.01149-14

    Figure Lengend Snippet: Loss of REST confers cell-type-specific phenotypes. (A and B) Relative absorbance of crystal violet stain representing cell numbers for MCF7 ( n = 24) (A) and MDA-MB-231 ( n = 18) (B) cells, expressed as a signal proportional to the REST norm , untreated

    Article Snippet: Although endogenous levels of IRS1 in MCF7 cells were reduced upon the introduction of IRS1-DN, there was little to no reduction in IRS1 levels in MDA-MB-231 cells.

    Techniques: Staining, Multiple Displacement Amplification

    Experimental setup and migration analysis. (A) Technical drawing of the μ -Slide Chemotaxis showing the three adjacent chambers for parallel experiments. Two reservoirs are connected by a central observation area (height: 70 μ m, width: 1 mm). Single cells are embedded in a 3D matrix inside the observation area. A time-stable gradient can be established by filling the reservoirs with different concentrations of chemoattractant (C 0 and C 100 ). (B) Representative image of the observation area with MDA-MB-231 cells embedded in a collagen gel with an overlay of the manually tracked cell trajectories at time point 24 h. Scale bar represents 500 μ m. (C) Each experimental setup includes three conditions: the experimental condition offering a concentration gradient (+/-) and the two controls with chemoattractant being present in the entire system (+/+) or without chemoattractant (-/-). (D) Analysis of chemotaxis by forward migration indices in direction of the gradient (FMI II ) and perpendicular to the gradient (FMI┴). FMI II values are high for directional migration and close to zero for random migration.

    Journal: PLoS ONE

    Article Title: Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

    doi: 10.1371/journal.pone.0203040

    Figure Lengend Snippet: Experimental setup and migration analysis. (A) Technical drawing of the μ -Slide Chemotaxis showing the three adjacent chambers for parallel experiments. Two reservoirs are connected by a central observation area (height: 70 μ m, width: 1 mm). Single cells are embedded in a 3D matrix inside the observation area. A time-stable gradient can be established by filling the reservoirs with different concentrations of chemoattractant (C 0 and C 100 ). (B) Representative image of the observation area with MDA-MB-231 cells embedded in a collagen gel with an overlay of the manually tracked cell trajectories at time point 24 h. Scale bar represents 500 μ m. (C) Each experimental setup includes three conditions: the experimental condition offering a concentration gradient (+/-) and the two controls with chemoattractant being present in the entire system (+/+) or without chemoattractant (-/-). (D) Analysis of chemotaxis by forward migration indices in direction of the gradient (FMI II ) and perpendicular to the gradient (FMI┴). FMI II values are high for directional migration and close to zero for random migration.

    Article Snippet: MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2 .

    Techniques: Migration, Chemotaxis Assay, Multiple Displacement Amplification, Concentration Assay

    MDA-MB-231 cell migration in linear EGF gradients. Serum-free medium containing EGF in different concentrations (0.015–15 nM) was filled in one reservoir and pure serum-free medium in the other reservoir (EGF/-). (A) MDA-MB-231 cells were exposed to time-stable linear gradients of EGF (0.015–15 nM/mm). Cell migration was evaluated by (B) the forward migration indices in direction of the gradient (FMI II ) and (D) the cell speed. (C) Cell velocity in direction of the gradient (V II ) was used to calculate the dissociation constant K d . Significances are indicated by asterisks with * for 0.01

    Journal: PLoS ONE

    Article Title: Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

    doi: 10.1371/journal.pone.0203040

    Figure Lengend Snippet: MDA-MB-231 cell migration in linear EGF gradients. Serum-free medium containing EGF in different concentrations (0.015–15 nM) was filled in one reservoir and pure serum-free medium in the other reservoir (EGF/-). (A) MDA-MB-231 cells were exposed to time-stable linear gradients of EGF (0.015–15 nM/mm). Cell migration was evaluated by (B) the forward migration indices in direction of the gradient (FMI II ) and (D) the cell speed. (C) Cell velocity in direction of the gradient (V II ) was used to calculate the dissociation constant K d . Significances are indicated by asterisks with * for 0.01

    Article Snippet: MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2 .

    Techniques: Multiple Displacement Amplification, Migration

    MDA-MB-231 cellular fitness and migration in standard DMEM/F-12 medium with different serum concentrations in comparison with serum-free growth medium UC. (A) Viability of MDA-MB-231 cells in chemoattractant-free medium. (B) Cell speed of MDA-MB-231 cells in chemoattractant-free medium. (C) Forward migration indices in direction of the gradient (FMI II ) and (D) perpendicular to the gradient (FMI ┴ ) of MDA-MB-231 cells migrating in pure medium (-/-), in a 1.5 nM/mm EGF gradient (EGF/-) and in 1.5 nM EGF in the entire chamber (EGF/EGF). Significances are indicated by asterisks with * for 0.01

    Journal: PLoS ONE

    Article Title: Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

    doi: 10.1371/journal.pone.0203040

    Figure Lengend Snippet: MDA-MB-231 cellular fitness and migration in standard DMEM/F-12 medium with different serum concentrations in comparison with serum-free growth medium UC. (A) Viability of MDA-MB-231 cells in chemoattractant-free medium. (B) Cell speed of MDA-MB-231 cells in chemoattractant-free medium. (C) Forward migration indices in direction of the gradient (FMI II ) and (D) perpendicular to the gradient (FMI ┴ ) of MDA-MB-231 cells migrating in pure medium (-/-), in a 1.5 nM/mm EGF gradient (EGF/-) and in 1.5 nM EGF in the entire chamber (EGF/EGF). Significances are indicated by asterisks with * for 0.01

    Article Snippet: MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2 .

    Techniques: Multiple Displacement Amplification, Migration

    Synergistic inhibition of MDA-MB-231 cell migration in a 1.5 nM/mm EGF gradient. The effect of the combination of EGFR inhibitors AG1478 (as TKI) and monoclonal anti-EGFR antibody 225 (as mAb) on migration was evaluated by the (A) forward migration indices in direction of the gradient (FMI II ) and (B) the cell speed. Four different combinations of AG1478 and anti-EGFR antibody were tested (white bars). Results were compared with the respective negative control without EGF gradient and gradient control without inhibitors (dark gray bars), as well as with both inhibitors applied separately (light gray bars). Significances are indicated by asterisks with * for 0.01

    Journal: PLoS ONE

    Article Title: Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

    doi: 10.1371/journal.pone.0203040

    Figure Lengend Snippet: Synergistic inhibition of MDA-MB-231 cell migration in a 1.5 nM/mm EGF gradient. The effect of the combination of EGFR inhibitors AG1478 (as TKI) and monoclonal anti-EGFR antibody 225 (as mAb) on migration was evaluated by the (A) forward migration indices in direction of the gradient (FMI II ) and (B) the cell speed. Four different combinations of AG1478 and anti-EGFR antibody were tested (white bars). Results were compared with the respective negative control without EGF gradient and gradient control without inhibitors (dark gray bars), as well as with both inhibitors applied separately (light gray bars). Significances are indicated by asterisks with * for 0.01

    Article Snippet: MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2 .

    Techniques: Inhibition, Multiple Displacement Amplification, Migration, Negative Control

    Inhibition of MDA-MB-231 cell migration in a 1.5 nM/mm EGF gradient. The effect of different EGFR inhibitors on migration was evaluated by the forward migration indices in direction of the gradient (FMI II ) and the cell speed, and compared to the respective negative control without EGF gradient (ctrl). (A) FMI II and (B) speed using tyrosine kinase inhibitor AG1478 (as TKI) (0.2 nM–20 μ M). (C) FMI II and (D) speed using monoclonal anti-EGFR antibody (as mAb) (0.5 ng/ml–5 μ g/ml). Significances are indicated by asterisks with * for 0.01

    Journal: PLoS ONE

    Article Title: Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

    doi: 10.1371/journal.pone.0203040

    Figure Lengend Snippet: Inhibition of MDA-MB-231 cell migration in a 1.5 nM/mm EGF gradient. The effect of different EGFR inhibitors on migration was evaluated by the forward migration indices in direction of the gradient (FMI II ) and the cell speed, and compared to the respective negative control without EGF gradient (ctrl). (A) FMI II and (B) speed using tyrosine kinase inhibitor AG1478 (as TKI) (0.2 nM–20 μ M). (C) FMI II and (D) speed using monoclonal anti-EGFR antibody (as mAb) (0.5 ng/ml–5 μ g/ml). Significances are indicated by asterisks with * for 0.01

    Article Snippet: MDA-MB-231 cells (DSMZ, DSMZ no. ACC-732) were maintained in the basal medium Dulbecco’s Modified Eagle`s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 37 °C in a humidified incubator at 5% CO2 .

    Techniques: Inhibition, Multiple Displacement Amplification, Migration, Negative Control

    Influence of TGF-β1 on TNBC cell signal transduction pathways. (A) MDA-MB-231 cells were treated with 5 ng/ml TGF-β1 and then analyzed by western blot analysis. Smad2 protein and phosphorylation of Smad2 protein, P38 protein and phosphorylation

    Journal: Oncology Letters

    Article Title: Association between transforming growth factor-β1 expression and the clinical features of triple negative breast cancer

    doi: 10.3892/ol.2016.4497

    Figure Lengend Snippet: Influence of TGF-β1 on TNBC cell signal transduction pathways. (A) MDA-MB-231 cells were treated with 5 ng/ml TGF-β1 and then analyzed by western blot analysis. Smad2 protein and phosphorylation of Smad2 protein, P38 protein and phosphorylation

    Article Snippet: MDA-MB-231 cells were purchased from Boster Biological Technology, Ltd., seeded at a density of 1×106 and cultured with L-15 culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO2 .

    Techniques: Transduction, Multiple Displacement Amplification, Western Blot

    Effect of TGF-β1 treatment on MDA-MB-231 invasion ability and migration

    Journal: Oncology Letters

    Article Title: Association between transforming growth factor-β1 expression and the clinical features of triple negative breast cancer

    doi: 10.3892/ol.2016.4497

    Figure Lengend Snippet: Effect of TGF-β1 treatment on MDA-MB-231 invasion ability and migration

    Article Snippet: MDA-MB-231 cells were purchased from Boster Biological Technology, Ltd., seeded at a density of 1×106 and cultured with L-15 culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO2 .

    Techniques: Multiple Displacement Amplification, Migration

    Macrophages associate with developing xenograft vessels. (A-G) Still images showing an MDA-MB-231 xenograft (A-D, Movie 3 ) or a B16-F1 xenograft (E-G, Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). Individual macrophages associated with the angiogenic region (yellow dashed circle) are indicated with yellow arrowheads, while macrophages associated with the control region (cyan dashed circle) are indicated with cyan arrowheads. The tumour region is outlined by a white dashed line in A and E. (H) Schematic demonstrating the positioning of the 10-μm angiogenic region (dashed yellow outline) at the tip of the blood vessel (green) and the control 10-μm control region (dashed cyan outline). (I) Quantitation of the percentage of frames during which a macrophage was observed at either an angiogenic region or a control region in MDA-MB-231 and B16-F1 xenografts, n =3. (J) Mean number of macrophages observed during each frame, in either the angiogenic region or the control region, in MDA-MB-231 and B16 F1 xenografts, n =3. (K-R) Still images showing a B16-F1 xenograft ( Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). An individual macrophage (yellow arrowhead) is tracked for 1 h 10 min during (K-N) and after (O-R) associating with the distal tip of a growing vessel (indicated by a cyan arrowhead). (S,T) Macrophage migration tracks of 12 macrophages during their period of contact with the tip of a growing vessel (S) and of the same macrophages once they leave the vessel tip (T). Each macrophage is depicted with the same colour in both S and T and they were tracked for identical periods of time during contact and post-contact. (U) Quantitation of macrophage migration speed during and after the period of contact with the tip of a growing vessel, n =12. Error bars represent s.d. * P

    Journal: Disease Models & Mechanisms

    Article Title: Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model

    doi: 10.1242/dmm.035998

    Figure Lengend Snippet: Macrophages associate with developing xenograft vessels. (A-G) Still images showing an MDA-MB-231 xenograft (A-D, Movie 3 ) or a B16-F1 xenograft (E-G, Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). Individual macrophages associated with the angiogenic region (yellow dashed circle) are indicated with yellow arrowheads, while macrophages associated with the control region (cyan dashed circle) are indicated with cyan arrowheads. The tumour region is outlined by a white dashed line in A and E. (H) Schematic demonstrating the positioning of the 10-μm angiogenic region (dashed yellow outline) at the tip of the blood vessel (green) and the control 10-μm control region (dashed cyan outline). (I) Quantitation of the percentage of frames during which a macrophage was observed at either an angiogenic region or a control region in MDA-MB-231 and B16-F1 xenografts, n =3. (J) Mean number of macrophages observed during each frame, in either the angiogenic region or the control region, in MDA-MB-231 and B16 F1 xenografts, n =3. (K-R) Still images showing a B16-F1 xenograft ( Movie 4 ) in an embryo with mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green). An individual macrophage (yellow arrowhead) is tracked for 1 h 10 min during (K-N) and after (O-R) associating with the distal tip of a growing vessel (indicated by a cyan arrowhead). (S,T) Macrophage migration tracks of 12 macrophages during their period of contact with the tip of a growing vessel (S) and of the same macrophages once they leave the vessel tip (T). Each macrophage is depicted with the same colour in both S and T and they were tracked for identical periods of time during contact and post-contact. (U) Quantitation of macrophage migration speed during and after the period of contact with the tip of a growing vessel, n =12. Error bars represent s.d. * P

    Article Snippet: B16-F1 and HEK-293T were originally obtained from the ATCC, while MDA-MB-231 cells were obtained from Caliper Life Sciences.

    Techniques: Multiple Displacement Amplification, Expressing, Quantitation Assay, Migration

    Macrophages are required for effective vegfaa -driven angiogenesis. (A-D) Confocal images taken at 2 dpi of kdrl:EGFP -expressing vessels (green) in zebrafish embryos implanted with either HEK-293T (A,B) or MDA-MB-231 (C,D) xenografts (white dashed line) transfected with either a control expression vector (A,C) or a vegfaa -expression vector (B,D). (E) Quantitation of graft vascularisation at 2 dpi, n > 17. (F-M) Confocal images taken at 2 dpi of mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green) in zebrafish embryos implanted with either HEK-293T or MDA-MB-231 xenografts (white dashed lines) transfected with either a control expression vector (F,H,J,L) or a vegfaa -expression vector (G,I,K,M) that have been injected with either PBS-containing liposomes (F-I) or clodronate-containing liposomes (J-M). (N) Quantitation of graft vascularisation at 2 dpi in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 16. (O) Schematic demonstrating how the proximal, middle and distal sections (marked by dashed red lines) of the xenograft (blue) were determined by their location with respect to the CCV (green). (P) Quantitation of vascularisation at 2 dpi in the proximal, middle and distal regions of vegfaa -expressing HEK-293T or MDA-MB-231 xenografts implanted into embryos injected with either PBS-containing or clodronate-containing liposomes, n > 19. Error bars represent s.d. n.s, P > 0.05; * P

    Journal: Disease Models & Mechanisms

    Article Title: Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model

    doi: 10.1242/dmm.035998

    Figure Lengend Snippet: Macrophages are required for effective vegfaa -driven angiogenesis. (A-D) Confocal images taken at 2 dpi of kdrl:EGFP -expressing vessels (green) in zebrafish embryos implanted with either HEK-293T (A,B) or MDA-MB-231 (C,D) xenografts (white dashed line) transfected with either a control expression vector (A,C) or a vegfaa -expression vector (B,D). (E) Quantitation of graft vascularisation at 2 dpi, n > 17. (F-M) Confocal images taken at 2 dpi of mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green) in zebrafish embryos implanted with either HEK-293T or MDA-MB-231 xenografts (white dashed lines) transfected with either a control expression vector (F,H,J,L) or a vegfaa -expression vector (G,I,K,M) that have been injected with either PBS-containing liposomes (F-I) or clodronate-containing liposomes (J-M). (N) Quantitation of graft vascularisation at 2 dpi in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 16. (O) Schematic demonstrating how the proximal, middle and distal sections (marked by dashed red lines) of the xenograft (blue) were determined by their location with respect to the CCV (green). (P) Quantitation of vascularisation at 2 dpi in the proximal, middle and distal regions of vegfaa -expressing HEK-293T or MDA-MB-231 xenografts implanted into embryos injected with either PBS-containing or clodronate-containing liposomes, n > 19. Error bars represent s.d. n.s, P > 0.05; * P

    Article Snippet: B16-F1 and HEK-293T were originally obtained from the ATCC, while MDA-MB-231 cells were obtained from Caliper Life Sciences.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Quantitation Assay, Injection

    Macrophages are not required for vascularisation in MDA-MB-231 xenografts depleted of VEGFA. (A) Quantitation of secreted VEGFA levels in 2×10 5 siRNA-treated cells, n =2. (B,C) Confocal images taken at 2 dpi of kdrl:EGFP -expressing vessels (green) in zebrafish embryos implanted with MDA-MB-231 xenografts (white dashed line) transfected with either control (B) or VEGFA siRNA (C). (D,E) Confocal images taken at 6 hpi of mpeg1:mCherry -expressing macrophages (red) and MDA-MB-231 xenografts (blue). (F,G) Quantitation of graft vascularisation at 2 dpi, n > 10 (F), and of graft-associated macrophages at 6, 24 and 48 hpi, n > 4 (G). (H) Quantitation of graft-associated macrophages at 6, 24 and 48 hpi, in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 5. (I-L) Confocal images taken at 2 dpi of mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green) in zebrafish embryos implanted with MDA-MB-231 xenografts (white dashed lines) transfected with either control (I,K) or VEGFA (J,L) siRNA that have been injected with either PBS-containing liposomes (I,J) or clodronate-containing liposomes (K,L). (M) Quantitation of graft vascularisation at 2 dpi in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 9. Error bars represent s.d. n.s, P > 0.05; * P

    Journal: Disease Models & Mechanisms

    Article Title: Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model

    doi: 10.1242/dmm.035998

    Figure Lengend Snippet: Macrophages are not required for vascularisation in MDA-MB-231 xenografts depleted of VEGFA. (A) Quantitation of secreted VEGFA levels in 2×10 5 siRNA-treated cells, n =2. (B,C) Confocal images taken at 2 dpi of kdrl:EGFP -expressing vessels (green) in zebrafish embryos implanted with MDA-MB-231 xenografts (white dashed line) transfected with either control (B) or VEGFA siRNA (C). (D,E) Confocal images taken at 6 hpi of mpeg1:mCherry -expressing macrophages (red) and MDA-MB-231 xenografts (blue). (F,G) Quantitation of graft vascularisation at 2 dpi, n > 10 (F), and of graft-associated macrophages at 6, 24 and 48 hpi, n > 4 (G). (H) Quantitation of graft-associated macrophages at 6, 24 and 48 hpi, in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 5. (I-L) Confocal images taken at 2 dpi of mpeg1:mCherry -expressing macrophages (red) and kdrl:EGFP -expressing blood vessels (green) in zebrafish embryos implanted with MDA-MB-231 xenografts (white dashed lines) transfected with either control (I,K) or VEGFA (J,L) siRNA that have been injected with either PBS-containing liposomes (I,J) or clodronate-containing liposomes (K,L). (M) Quantitation of graft vascularisation at 2 dpi in embryos injected with either PBS-containing or clodronate-containing liposomes, n > 9. Error bars represent s.d. n.s, P > 0.05; * P

    Article Snippet: B16-F1 and HEK-293T were originally obtained from the ATCC, while MDA-MB-231 cells were obtained from Caliper Life Sciences.

    Techniques: Multiple Displacement Amplification, Quantitation Assay, Expressing, Transfection, Injection

    Role of MEK/ERK signaling pathway in migration induced by refp17 and vp17s. MDA-MB 231 cells were serum starved for 24 h in the presence or absence of the PI3K/Akt inhibitor LY294002 (20 μM), the Jak/STAT inhibitor AG-490 (20 μM), or the MEK/ERK1/2 inhibitor PD98059 (10 μM). a Confluent cell monolayers were serum starved for 24 h and then scratched with a 200 μl pipette tip. Cells were then incubated for 6 h in the absence (NT) or in the presence of 10 ng/ml of refp17 or vp17s. Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (*** p

    Journal: Infectious Agents and Cancer

    Article Title: HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

    doi: 10.1186/s13027-017-0160-7

    Figure Lengend Snippet: Role of MEK/ERK signaling pathway in migration induced by refp17 and vp17s. MDA-MB 231 cells were serum starved for 24 h in the presence or absence of the PI3K/Akt inhibitor LY294002 (20 μM), the Jak/STAT inhibitor AG-490 (20 μM), or the MEK/ERK1/2 inhibitor PD98059 (10 μM). a Confluent cell monolayers were serum starved for 24 h and then scratched with a 200 μl pipette tip. Cells were then incubated for 6 h in the absence (NT) or in the presence of 10 ng/ml of refp17 or vp17s. Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (*** p

    Article Snippet: In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways.

    Techniques: Migration, Multiple Displacement Amplification, Transferring, Incubation

    Effects of refp17 and vp17s on Akt, STAT3 and ERK1/2 activity in MDA-MB 231 cells. Cells were treated or not (NT) for 30 min with 50, 100 and 200 ng/ml of refp17 or vp17s and then lysed. Equal amounts of total cellular extracts were analyzed for expression of pAkt, Akt, pSTAT3, STAT3, pERK1/2 or ERK1/2 by western blot analysis using mAbs to pAkt (Ser473), Akt, pSTAT3 (Tyr705), STAT3, pERK1/2 (Thr202, Tyr204) or ERK1/2 as specific reagents. Phosphorylation of Akt, STAT3 and ERK1/2 was verified by densiometric analysis and plotting of the pAkt/Akt, pSTAT3/STAT3 and pERK1/2/ERK1/2. Upper panel, Blots from one representative experiment of three with similar results are shown. Lower panel, Values reported for Akt, STAT3 and ERK1/2 are the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (** p

    Journal: Infectious Agents and Cancer

    Article Title: HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

    doi: 10.1186/s13027-017-0160-7

    Figure Lengend Snippet: Effects of refp17 and vp17s on Akt, STAT3 and ERK1/2 activity in MDA-MB 231 cells. Cells were treated or not (NT) for 30 min with 50, 100 and 200 ng/ml of refp17 or vp17s and then lysed. Equal amounts of total cellular extracts were analyzed for expression of pAkt, Akt, pSTAT3, STAT3, pERK1/2 or ERK1/2 by western blot analysis using mAbs to pAkt (Ser473), Akt, pSTAT3 (Tyr705), STAT3, pERK1/2 (Thr202, Tyr204) or ERK1/2 as specific reagents. Phosphorylation of Akt, STAT3 and ERK1/2 was verified by densiometric analysis and plotting of the pAkt/Akt, pSTAT3/STAT3 and pERK1/2/ERK1/2. Upper panel, Blots from one representative experiment of three with similar results are shown. Lower panel, Values reported for Akt, STAT3 and ERK1/2 are the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (** p

    Article Snippet: In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways.

    Techniques: Activity Assay, Multiple Displacement Amplification, Expressing, Western Blot

    Refp17 and vp17s promote breast cancer cells migration. In the wound-healing assay, confluent MDA-MB 231 cell monolayers were serum starved for 24 h and then scratched using a 200 μl pipette tip. Cells were cultured in complete medium either unsupplemented or containing 10 ng/ml of refp17 or vp17s. a After 6 h of culture cells were fixed and stained with Coomassie brilliant blue. The cells migrated into the wound area were counted. b The percentage of wound healing was observed over a period of 12 h. c Wound healing assay was performed pretreating MDA-MB 231 cells for 1 h with a neutralizing mAb to CXCR1 (2.5 μg/ml), CXCR2 (2.5 μg/ml), or with an isotype-matched mAb (Ctrl mAb; 2.5 μg/ml). Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (** p

    Journal: Infectious Agents and Cancer

    Article Title: HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

    doi: 10.1186/s13027-017-0160-7

    Figure Lengend Snippet: Refp17 and vp17s promote breast cancer cells migration. In the wound-healing assay, confluent MDA-MB 231 cell monolayers were serum starved for 24 h and then scratched using a 200 μl pipette tip. Cells were cultured in complete medium either unsupplemented or containing 10 ng/ml of refp17 or vp17s. a After 6 h of culture cells were fixed and stained with Coomassie brilliant blue. The cells migrated into the wound area were counted. b The percentage of wound healing was observed over a period of 12 h. c Wound healing assay was performed pretreating MDA-MB 231 cells for 1 h with a neutralizing mAb to CXCR1 (2.5 μg/ml), CXCR2 (2.5 μg/ml), or with an isotype-matched mAb (Ctrl mAb; 2.5 μg/ml). Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (** p

    Article Snippet: In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways.

    Techniques: Migration, Wound Healing Assay, Multiple Displacement Amplification, Transferring, Cell Culture, Staining

    Effects of refp17 and vp17s on viability and colony-forming capacity of MDA-MB 231 cells. a MTT assay was used to determine the viability of MDA-MB 231 cells treated or not for 48 h with refp17 or vp17s as indicated. Data represent the average of three independent experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data. b The effect of refp17 and vp17s on breast cancer cells clonogenicity was analyzed by soft agar assay. Cells were plated in six-well plate and, after two days, the medium was replaced using fresh medium with various concentration of refp17 or vp17s (range from 50 to 200 ng/ml). Not treated cells (NT) were used as a negative control. Data represent the average number of colonies ± SD from three independent experiments performed in triplicate. The statistical significance between control and treated cultures was calculated using two-way ANOVA, and the Bonferroni post-test was used to compare data (** p

    Journal: Infectious Agents and Cancer

    Article Title: HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

    doi: 10.1186/s13027-017-0160-7

    Figure Lengend Snippet: Effects of refp17 and vp17s on viability and colony-forming capacity of MDA-MB 231 cells. a MTT assay was used to determine the viability of MDA-MB 231 cells treated or not for 48 h with refp17 or vp17s as indicated. Data represent the average of three independent experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data. b The effect of refp17 and vp17s on breast cancer cells clonogenicity was analyzed by soft agar assay. Cells were plated in six-well plate and, after two days, the medium was replaced using fresh medium with various concentration of refp17 or vp17s (range from 50 to 200 ng/ml). Not treated cells (NT) were used as a negative control. Data represent the average number of colonies ± SD from three independent experiments performed in triplicate. The statistical significance between control and treated cultures was calculated using two-way ANOVA, and the Bonferroni post-test was used to compare data (** p

    Article Snippet: In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways.

    Techniques: Multiple Displacement Amplification, MTT Assay, Soft Agar Assay, Concentration Assay, Negative Control

    Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.

    Journal: Breast Cancer Research : BCR

    Article Title: Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin

    doi: 10.1186/bcr2586

    Figure Lengend Snippet: Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.

    Article Snippet: To support the effects observed after siRNA knockdown in Figures , , , , and as being AGR2-specific effects and not an off-target effect of the Invitrogen siRNA reagent, additional AGR2 siRNA reagents were used. siRNA reagents targeting distinct sequences from Ambion (Ambion Silencer) and Dharmacon (On-Target Plus Smartpool) were used in anchorage-dependent functional studies in T47 D and MDA-MB-231 cells with effects similar to those seen with the Invitrogen reagent (see Supplementary figure S1a in Additional file ).

    Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Multiple Displacement Amplification, MTT Assay

    AGR2 knockdown reduces cell proliferation in ER-positive breast cancer cells . Cell-cycle profiles were analyzed with BrdU incorporation. Cells were pulse-labeled with 10 μM BrdU 48 hours after transfection and analyzed for BrdU incorporation with FACS. Cells were gated on Sub G 1 , G 0 /G 1 , S, and G 2 /M populations. (a) T47 D, (b) ZR-75-1, (c) MDA-MB-231, and (d) SK-BR-3 cells.

    Journal: Breast Cancer Research : BCR

    Article Title: Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin

    doi: 10.1186/bcr2586

    Figure Lengend Snippet: AGR2 knockdown reduces cell proliferation in ER-positive breast cancer cells . Cell-cycle profiles were analyzed with BrdU incorporation. Cells were pulse-labeled with 10 μM BrdU 48 hours after transfection and analyzed for BrdU incorporation with FACS. Cells were gated on Sub G 1 , G 0 /G 1 , S, and G 2 /M populations. (a) T47 D, (b) ZR-75-1, (c) MDA-MB-231, and (d) SK-BR-3 cells.

    Article Snippet: To support the effects observed after siRNA knockdown in Figures , , , , and as being AGR2-specific effects and not an off-target effect of the Invitrogen siRNA reagent, additional AGR2 siRNA reagents were used. siRNA reagents targeting distinct sequences from Ambion (Ambion Silencer) and Dharmacon (On-Target Plus Smartpool) were used in anchorage-dependent functional studies in T47 D and MDA-MB-231 cells with effects similar to those seen with the Invitrogen reagent (see Supplementary figure S1a in Additional file ).

    Techniques: BrdU Incorporation Assay, Labeling, Transfection, FACS, Multiple Displacement Amplification

    siRNA-mediated AGR2 knockdown affects anchorage-dependent and anchorage-independent growth in breast cancer cell lines . T47 D, ZR-75-1, MDA-MB-231, and SK-BR-3 cells were transfected with negative control siRNA (iNC), AGR2 siRNA (iAGR2), or untransfected (UT). KSP (DKSP) and its corresponding control (DNC) were used as transfection controls. Results are expressed as a ratio of untransfected cells (±SD), n = 3. (a) Detection of endogenous AGR2 in breast cancer cell line supernatants by IP-Western and whole-cell lysates by Western. AGR2 knockdown was confirmed in lysates 72 hours after transfection. β-Actin served as a loading control. (b) The impact of iAGR2 on anchorage-dependent growth was evaluated at 96 hours after transfection by using the Cell Titer Glo assay. Anchorage-independent growth assays were also used: (i) soft agar colony formation assay (c) , with Alamar blue as a readout; (ii) spheroid assay (d) , in which lysed spheroid LDH levels were representative of total cell number after 8 days; corresponding spheroid images were also captured. * P

    Journal: Breast Cancer Research : BCR

    Article Title: Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin

    doi: 10.1186/bcr2586

    Figure Lengend Snippet: siRNA-mediated AGR2 knockdown affects anchorage-dependent and anchorage-independent growth in breast cancer cell lines . T47 D, ZR-75-1, MDA-MB-231, and SK-BR-3 cells were transfected with negative control siRNA (iNC), AGR2 siRNA (iAGR2), or untransfected (UT). KSP (DKSP) and its corresponding control (DNC) were used as transfection controls. Results are expressed as a ratio of untransfected cells (±SD), n = 3. (a) Detection of endogenous AGR2 in breast cancer cell line supernatants by IP-Western and whole-cell lysates by Western. AGR2 knockdown was confirmed in lysates 72 hours after transfection. β-Actin served as a loading control. (b) The impact of iAGR2 on anchorage-dependent growth was evaluated at 96 hours after transfection by using the Cell Titer Glo assay. Anchorage-independent growth assays were also used: (i) soft agar colony formation assay (c) , with Alamar blue as a readout; (ii) spheroid assay (d) , in which lysed spheroid LDH levels were representative of total cell number after 8 days; corresponding spheroid images were also captured. * P

    Article Snippet: To support the effects observed after siRNA knockdown in Figures , , , , and as being AGR2-specific effects and not an off-target effect of the Invitrogen siRNA reagent, additional AGR2 siRNA reagents were used. siRNA reagents targeting distinct sequences from Ambion (Ambion Silencer) and Dharmacon (On-Target Plus Smartpool) were used in anchorage-dependent functional studies in T47 D and MDA-MB-231 cells with effects similar to those seen with the Invitrogen reagent (see Supplementary figure S1a in Additional file ).

    Techniques: Multiple Displacement Amplification, Transfection, Negative Control, Western Blot, Glo Assay, Soft Agar Assay

    Olaparib attenuates PD-1 binding and T-cell-mediated cell death in TNBC cells (A) FACS analysis of cell surface PD-1 binding of MDA-MB-231 cells treated with 10 μM olaparib or 10 nM talazoparib for 24 hours. (B) FACS analysis of PARP1 knockdown (K/D), PARP1 knockout (K/O), and parental MDA-MB-231 cells. (C) SUM149 cells were treated with the indicated concentrations of olaparib for 10 days. (D) MDA-MB-231 cells expressing nuclear RFP protein were first treated with or without olaparib (10 μM) for 3 hours and then co-cultured with or without activated peripheral blood mononuclear cells (PBMCs). Left, quantitation showing the number of live cells per well, counting the number of red fluorescent objects, normalized to that at the zero time point. Right, the percent of T cell-meditated tumor cell killing observed at 72 hours in activated PBMC co-culture with control or olaparib-treated cells (normalized to co-culture without PBMCs). (E) Left, representative merged images showing red fluorescent (nuclear restricted RFP), and green fluorescent (Caspase 3/7 substrate) objects in MDA-MB-231 cells co-cultured with activated PBMCs at 0 and 72 hours. Images were taken using the IncuCyte Zoom microscope. Right, quantitation showing the number of live cells following treatment with olaparib (10 μM), PD-L1 antibody (PD-L1 Ab; 10 μg/ml), or the combination co-cultured with activated PBMCs for 72 hours. The number of live cells (red fluorescent objects) were counted and normalized to that at the zero time point. * P

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: PARP inhibitor upregulates PD-L1 expression and enhances cancer-associated immunosuppression

    doi: 10.1158/1078-0432.CCR-16-3215

    Figure Lengend Snippet: Olaparib attenuates PD-1 binding and T-cell-mediated cell death in TNBC cells (A) FACS analysis of cell surface PD-1 binding of MDA-MB-231 cells treated with 10 μM olaparib or 10 nM talazoparib for 24 hours. (B) FACS analysis of PARP1 knockdown (K/D), PARP1 knockout (K/O), and parental MDA-MB-231 cells. (C) SUM149 cells were treated with the indicated concentrations of olaparib for 10 days. (D) MDA-MB-231 cells expressing nuclear RFP protein were first treated with or without olaparib (10 μM) for 3 hours and then co-cultured with or without activated peripheral blood mononuclear cells (PBMCs). Left, quantitation showing the number of live cells per well, counting the number of red fluorescent objects, normalized to that at the zero time point. Right, the percent of T cell-meditated tumor cell killing observed at 72 hours in activated PBMC co-culture with control or olaparib-treated cells (normalized to co-culture without PBMCs). (E) Left, representative merged images showing red fluorescent (nuclear restricted RFP), and green fluorescent (Caspase 3/7 substrate) objects in MDA-MB-231 cells co-cultured with activated PBMCs at 0 and 72 hours. Images were taken using the IncuCyte Zoom microscope. Right, quantitation showing the number of live cells following treatment with olaparib (10 μM), PD-L1 antibody (PD-L1 Ab; 10 μg/ml), or the combination co-cultured with activated PBMCs for 72 hours. The number of live cells (red fluorescent objects) were counted and normalized to that at the zero time point. * P

    Article Snippet: NucLight RFP MDA-MB-231 cells (#4457, Essen Bioscience, Ann Arbor, MI) were seeded in a 96-well plate with or without olaparib.

    Techniques: Binding Assay, FACS, Multiple Displacement Amplification, Knock-Out, Expressing, Cell Culture, Quantitation Assay, Co-Culture Assay, Microscopy

    Flow cytometry analysis of apoptosis in MDA-MB-231 cells. a Representative images of flow cytometry. b Apoptotic rates in cells treated with the stated concentrations of BPTS. ** P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Total saponins of Bolbostemma paniculatum (maxim.) Franquet exert antitumor activity against MDA-MB-231 human breast cancer cells via inhibiting PI3K/Akt/mTOR pathway

    doi: 10.1186/s12906-019-2708-0

    Figure Lengend Snippet: Flow cytometry analysis of apoptosis in MDA-MB-231 cells. a Representative images of flow cytometry. b Apoptotic rates in cells treated with the stated concentrations of BPTS. ** P

    Article Snippet: Cell culture MDA-MB-231 cells were purchased from OBiO Technology Corp., Ltd. (Shanghai, China), and grown in L-15 supplemented with 10% FBS and 1% penicillin-streptomycin in a saturated humidity incubator (37 °C, 5%CO2 ).

    Techniques: Flow Cytometry, Cytometry, Multiple Displacement Amplification

    Protein expression levels of p-PI3K, p-Akt, p-mTOR in MDA-MB-231 cells treated with BPTS. ( A ) Western blot of the proteins in cells treated with BPTS alone, ( B ) BPTS + LY294002 and ( C ) BPTS + rapamycin. ( a ) Representative blot of expression. ( b ), ( c ) and ( d ) Quantification of expression normalized to expression of β-actin. * P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Total saponins of Bolbostemma paniculatum (maxim.) Franquet exert antitumor activity against MDA-MB-231 human breast cancer cells via inhibiting PI3K/Akt/mTOR pathway

    doi: 10.1186/s12906-019-2708-0

    Figure Lengend Snippet: Protein expression levels of p-PI3K, p-Akt, p-mTOR in MDA-MB-231 cells treated with BPTS. ( A ) Western blot of the proteins in cells treated with BPTS alone, ( B ) BPTS + LY294002 and ( C ) BPTS + rapamycin. ( a ) Representative blot of expression. ( b ), ( c ) and ( d ) Quantification of expression normalized to expression of β-actin. * P

    Article Snippet: Cell culture MDA-MB-231 cells were purchased from OBiO Technology Corp., Ltd. (Shanghai, China), and grown in L-15 supplemented with 10% FBS and 1% penicillin-streptomycin in a saturated humidity incubator (37 °C, 5%CO2 ).

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot

    Effect of BPTS on proliferation of MDA-MB-231 cells. Results are presented as a percentage of the 0 μg/mL BPTS group. * P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Total saponins of Bolbostemma paniculatum (maxim.) Franquet exert antitumor activity against MDA-MB-231 human breast cancer cells via inhibiting PI3K/Akt/mTOR pathway

    doi: 10.1186/s12906-019-2708-0

    Figure Lengend Snippet: Effect of BPTS on proliferation of MDA-MB-231 cells. Results are presented as a percentage of the 0 μg/mL BPTS group. * P

    Article Snippet: Cell culture MDA-MB-231 cells were purchased from OBiO Technology Corp., Ltd. (Shanghai, China), and grown in L-15 supplemented with 10% FBS and 1% penicillin-streptomycin in a saturated humidity incubator (37 °C, 5%CO2 ).

    Techniques: Multiple Displacement Amplification