Journal: Oncogenesis

Article Title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ

doi: 10.1038/oncsis.2016.10

Figure Lengend Snippet: Effect of Bag-1L overexpression on MCF-10A 2D cultures. Bag-1 protein expression was examined in MCF-10A clones grown in 2D cultures as indicated by: ( a ) Immunoblot where β-actin was included as a control for loading and ( b ) immunocytochemical labeling for Bag-1 (red) and nuclear counterstain DAPI (blue); two exposure times are shown for Bag-1 staining; scale bars=50 μm. ( c ) Phase-contrast images reveal that Bag-1L-overexpressing clones acquire a cobblestone morphology, which is typical of MCF-10A cells; scale bars=200 μm. ( d ) The colony-forming efficiency of Bag-1L clones was assessed in a clonogenic assay and expressed as the plating efficiency. Values represent the mean±s.e.m. from three independent experiments, each with three technical replicates. ( e ) Proliferation of clones was measured at days 2 and 4 of culture. Cells (20 000/well) were plated at day 0 and were fixed in methanol and stained with crystal violet. Stain was dissolved in 20% acetic acid and absorbance at 595 nm recorded. Bar graphs represent the mean±s.e.m. values from three independent experiments, each with three technical replicates. * P <0.05, ** P <0.01 as determined by two-way ANOVA with Bonferroni's multiple comparisons test.

Article Snippet: MCF-10A cells were obtained from LGC Standards (Teddington, UK) and were cultured and maintained as described by Debnath et al. To generate cell clones, pcDNA3-Bag-1L or empty pcDNA3 vectors were transfected into MCF-10A cells using FuGene HD (Promega, Southampton, UK) and stable integrants selected with Geneticin (100 μg/ml; Sigma, Gillingham, UK).

Techniques: Over Expression, Expressing, Clone Assay, Western Blot, Labeling, Staining, Clonogenic Assay

Journal: Oncogenesis

Article Title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ

doi: 10.1038/oncsis.2016.10

Figure Lengend Snippet: Bag-1L overexpression attenuates luminal clearing and promotes an abnormal acinar morphology. ( a ) Immunoblot analysis shows expression of Bag-1 isoforms in lysates from MCF-10A acini clones cultured for 12 days; β-actin was included as a loading control. Densitometric analysis shows the abundance of Bag-1 S and Bag-1L protein isoforms relative to pcDNA control. ( b ) Representative confocal immunofluorescence images taken through the center of MCF-10A acini at day 20 of culture. Cells were stained with phalloidin-TRITC (green) and nuclei counterstained with DAPI (red); scale bars=100 μm. ( c ) Acini with filled lumens were counted at day 20 of culture and their number is expressed as a percentage of total acini. ( d ) Representative phase-contrast images of MCF-10A cell clones cultured in 3D for 20 days revealing gross external morphology; white scale bars=500 μm; black scale bars=200 μm. ( e ) Acini with abnormal morphology were counted at day 20 of culture and their number is expressed as a percentage of total acini. Values represent the mean±s.e.m. from at least three independent experiments, each with two technical replicates. * P <0.05, ** P <0.01, **** P <0.0001 as determined by two-way ANOVA with Bonferroni's multiple comparisons test.

Article Snippet: MCF-10A cells were obtained from LGC Standards (Teddington, UK) and were cultured and maintained as described by Debnath et al. To generate cell clones, pcDNA3-Bag-1L or empty pcDNA3 vectors were transfected into MCF-10A cells using FuGene HD (Promega, Southampton, UK) and stable integrants selected with Geneticin (100 μg/ml; Sigma, Gillingham, UK).

Techniques: Over Expression, Western Blot, Expressing, Clone Assay, Cell Culture, Immunofluorescence, Staining

Journal: Oncogenesis

Article Title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ

doi: 10.1038/oncsis.2016.10

Figure Lengend Snippet: Bag-1L overexpression does not prevent cell cycle arrest but attenuates luminal apoptosis during acini morphogenesis. Representative confocal immunofluorescence images taken through the center of MCF-10A acini at the indicated time points of 3D culture. ( a ) Proliferation was examined using Ki67 (green) as a marker. ( b ) Apoptosis was assessed using M30 (green) as a marker of caspase-cleaved cytokeratin 18. Nuclei were counterstained with DAPI (red); scale bars=50 μm.

Article Snippet: MCF-10A cells were obtained from LGC Standards (Teddington, UK) and were cultured and maintained as described by Debnath et al. To generate cell clones, pcDNA3-Bag-1L or empty pcDNA3 vectors were transfected into MCF-10A cells using FuGene HD (Promega, Southampton, UK) and stable integrants selected with Geneticin (100 μg/ml; Sigma, Gillingham, UK).

Techniques: Over Expression, Immunofluorescence, Marker

Journal: Oncogenesis

Article Title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ

doi: 10.1038/oncsis.2016.10

Figure Lengend Snippet: HER2 overexpression promotes atypical MCF-10A morphology in 3D and insulin unresponsiveness in 2D cultures. ( a ) Immunoblot analysis of HER2 overexpression in retrovirally transduced MCF-10A-pooled clones grown in 2D culture; β-actin was included as a control for loading. ( b ) Immunofluorescence staining for HER2 (red) in 2D cultures of MCF-10A parental or retrovirally transduced with pBabe-puro vector control (puro) or pBabe-puro/(HER2); nuclei were counterstained with DAPI (blue), whereas secondary antibody alone was used to exclude non-specific staining; scale bars=50 μm. ( c ) Representative confocal immunofluorescence images of MCF-10A acini at day 12 of culture. Acini were stained with phalloidin-TRITC (green) and nuclei counterstained with DAPI (red); scale bars=50 μm. The number of acini with ( d ) filled lumens (day 12) or ( e ) abnormal morphology (day 20) was counted and is expressed as a percentage of the total number of acini. Values represent the mean±s.e.m. from three independent experiments, each with four technical replicates. ** P <0.01, **** P <0.0001 as determined by two-way ANOVA with Bonferroni's multiple comparisons test. ( f ) Insulin and EGF sensitivity was assessed in 2D culture under serum-free conditions (24 h serum starvation) following treatment with the indicated growth factors for 48 h. Bar graphs represent the mean fold change±s.e.m. in absorbance (595 nm), corresponding to cell growth, in growth factor-supplemented relative to growth factor-free media for each cell line, which was determined by crystal violet assay. Data are from at least three independent experiments, each with three technical replicates. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 as determined by two-way ANOVA with Bonferroni's multiple comparisons test.

Article Snippet: MCF-10A cells were obtained from LGC Standards (Teddington, UK) and were cultured and maintained as described by Debnath et al. To generate cell clones, pcDNA3-Bag-1L or empty pcDNA3 vectors were transfected into MCF-10A cells using FuGene HD (Promega, Southampton, UK) and stable integrants selected with Geneticin (100 μg/ml; Sigma, Gillingham, UK).

Techniques: Over Expression, Western Blot, Clone Assay, Immunofluorescence, Staining, Transduction, Plasmid Preparation, Crystal Violet Assay

Journal: Oncogenesis

Article Title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ

doi: 10.1038/oncsis.2016.10

Figure Lengend Snippet: Thio-2 can partially reverse the abnormal acinar morphology associated with Bag-1L overexpression. ( a ) MCF-10A cells were treated with Thioflavin S or Thio-2 at the indicated concentrations for 5 days and viability was assessed by CellTiter Aqueous One solution assay relative to DMSO-treated cells at each concentration. Values represent the mean±s.d. as percentage of DMSO control from three experiments, each with three technical replicates. **** P <0.0001 was determined by two-way ANOVA with Bonferroni's multiple comparisons test relative to the effect of the lowest concentration of Thio-2 on each corresponding cell type. ( b ) MCF-10A cells were treated for 24 h with Thio-2 (50 μ M ) or DMSO (0.5% v/v) before seeding on Matrigel and acini were allowed to form over 14 days; further treatment (25 μ M Thio-2 or 0.25% v/v DMSO) was administered on days 4 and 8 of 3D culture. Acini exhibiting abnormal morphology were quantified and expressed as a percentage of the total number. Values represent the mean±s.e.m. from four independent experiments, each with two technical replicates. * P <0.05, ** P <0.01, *** P <0.001 as determined by two-way ANOVA with Bonferroni's multiple comparisons test. ( c ) Representative phase-contrast microscopy images show the atypical branching external morphology of Bag-1L/B acini in the presence of DMSO or Thio-2; scale bars=50 μm. ( d ) Immunoblot analysis shows the effect of serum (10%) stimulation alone or in the presence of DMSO (0.5%), Thio-2 (50 μ M ) or U0126 (25 μ M ) on the activation of ERK and AKT in serum-deprived cells; β-actin was used as a loading control.

Article Snippet: MCF-10A cells were obtained from LGC Standards (Teddington, UK) and were cultured and maintained as described by Debnath et al. To generate cell clones, pcDNA3-Bag-1L or empty pcDNA3 vectors were transfected into MCF-10A cells using FuGene HD (Promega, Southampton, UK) and stable integrants selected with Geneticin (100 μg/ml; Sigma, Gillingham, UK).

Techniques: Over Expression, Concentration Assay, Microscopy, Western Blot, Activation Assay

Journal: Oncology Reports

Article Title: BKCa channel inhibitor modulates the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway

doi: 10.3892/or.2014.3617

Figure Lengend Snippet: BKCa channels regulate the transmembrane potential in breast cancer cells. (A) Breast cancer cells displayed higher levels of BKCa transcripts compared to benign mammary epithelial cells. Total RNA from MCF10A benign mammary epithelial cells and UACC893, MDA-MB-231 and SK-BR-3 beast cancer cells was assayed for BKCa transcript levels using quantitative RT-PCR. Statistically significant differences vs. the MCF10A cells. N=3, P≤0.05. (B) BKCa channel proteins (green) were detected in UACC893 (upper panels), MDA-MB-231 (middle panels) and SK-BR-3 (lower panels) cells using fluorescent immunocytochemistry. Nuclei were counterstained with propidium iodide (red) and the image series were acquired using Zeiss 510 laser confocal microscope. A series was reconstructed to generate three dimensional images rotated to present the lateral sides of the cells with apical surfaces facing atop of their respective image. MDA-MB-231 cells did not possess basal-apical differentiation and appear flat in the images. Experiments were repeated at least three times for each cell line. (C) IbTX differentially modulates transmembrane potential. UACC893 (upper panel), MDA-MB-231 (middle panel) and SK-BR-3 (lower panel) cells pre-loaded with 2 μmol/l DiBac4(3) in membrane potential buffer and supplemented with 10 nmol/l IbTX were observed using a Zeiss 510 laser confocal microscope for 20 min with images captured every minute. Average signal intensities from 5 to 7 regions of interest were calculated and plotted. Increased signal intensity signifies depolarization due to IbTX inhibition of BKCa channels by IbTX. Significant differences vs. start of experiment (1 min). N=3. IbTX, iberiotoxin.

Article Snippet: UACC893, MDA-MB-231, SK-BR-3 and MCF10A cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Immunocytochemistry, Microscopy, Membrane, Inhibition