aib1 src  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: ( A ) The experimental paradigm. The differential responses to estradiol (E2) treatment of MCF-7 (cell growth) and long-term estrogen deprived MCF-7:5C cells (apoptosis) are indicated. Proteomics profiles of the two cell lines at baseline and after a brief (2 h) E2 treatment were generated using immunoprecipitations (IP). Proteins interacting with AIB1 or phosphotyrosine containing protein complexes were isolated by IP followed by mass spectrometry. Data were then subjected to an integrated bioinformatics analysis of signaling pathways and protein networks. ( B,C ) Reversal of E2-dependent effects on MCF-7 and MCF-7:5C after depletion of endogenous AIB1 protein using two different lentiviral shRNAs. MCF-7 or MCF-7:5C cells were infected with lentiviral particles expressing control or AIB1-targeting shRNAs. (B) RNAi-mediated knockdown was assayed by Western blot analysis for AIB1 relative to an actin loading control. (C) Cell growth was assayed 6 days after plating without or with E2. The E2 effect is shown relative to the respective untreated controls (mean ±S.E.M.). Closed circles: control shRNA; Open circles (red): AIB1 shRNA. #, p<0.05 E2 treatment effect vs. no treatment in control shRNA cells; *, p<0.05 E2 treatment effect in control shRNA cells vs. E2 treatment in AIB1 depleted cells. Representative data from one of at least three independent experiments are shown.

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques: Generated, Isolation, Mass Spectrometry, Infection, Expressing, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: Venn diagrams of proteins identified from anti-AIB1 ( A,C ) or anti-pY IP ( B,D ) experimental groups. ( C,D ) Proteins in combined AIB1-IP or pY-IP data sets. Individual proteins and subgroups are shown in & .

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: The thick grey line in the middle provides an arbitrary boundary between the pathways. Anti-AIB1 immunoprecipitated (AIB1-IPed) and anti-pY-immunoprecipitated proteins (pY-IPed) are indicated by red or green circles respectively (keys at the bottom). The blue circled proteins are AIB1-IPed proteins from MCF-7 (CALM1) or MCF-7:5C cells (β-catenin) under both E2-treated and untreated conditions; the purple circled one (ITPR3) is an AIB1-IPed protein from both cells only under E2 treated condition, while the yellow circled one (TYK2) is an AIB1-IPed protein from both cells under both E2 treated and untreated conditions. Proteins circled in grey are from known canonical pathways (e.g. ERK in cell growth or BAD in apoptosis) but not identified here. Solid line arrows indicate direct interactions (e.g. CDK1 phosphorylates Rap1GAP) or translocations (e.g. catenins) of proteins, while dashed arrows indicate indirect actions of proteins (e.g. AKT activate MEK through several steps). Hammer-ended lines indicate inhibitory effects on the target. Detailled pathways are shown in , , , .

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Downregulation of Steroid Receptor Coactivator-2 Modulates Estrogen-Responsive Genes and Stimulates Proliferation of MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0070096

Figure Lengend Snippet: (A). Quantification of SRC-2 mRNA expression in shRNA lentivirus-infected MCF-7 cells. mRNA levels of SRC-2 in a MCF-7 cell line infected with shRNA targeting SRC-2 (SRC-2 shRNA) were compared to the expression in a control shRNA MCF-7 cell line (Ctr shRNA), and in a MCF-7 cell line transduced with shRNA lentivirus targeting SRC-3 (SRC-3 shRNA). The mRNA expression of SRC-2 is relative to TBP mRNA. The results are representative of at least three independent experiments. (B). Western blotting analyses of SRC-2-depleted MCF-7 cells. MCF-7 cells infected with shRNA lentivirus targeting SRC-2 (SRC-2 shRNA) or a negative control shRNA empty vector (Ctr shRNA), were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The Ctr shRNA cells were then treated with either Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. Immunoblotting was performed with anti-TIF2 antibody and anti-GAPDH antibody. The results shown are representative of at least three independent experiments. (C). Microarray analyses of five RNA samples isolated from five individual cell samples of shRNA control MCF-7 cells (Ctr shRNA), SRC-2 KD MCF-7 cells (SRC-2 shRNA) and control shRNA cells treated with cAMP elevating agents, as described in A. A Venn diagram shows the number of individual and overlapping sets of genes differentially expressed after SRC-2 KD (SRC-2 shRNA) and after treatment with cAMP elevating agents. To examine which genes were similarly differentially expressed between the two treated groups when compared to control, a SAM analysis with overlapping genes was performed. The fold change cut-off value ≥1.5, and q-value = 0, was used to determine differentially expressed genes.

Article Snippet: Five individual lentiviral pLKO.1-puro short hairpin (sh) RNA plasmids targeting different sequences on SRC-2/GRIP1 or SRC-3/AIB1 mRNA and a pLKO.1-puro empty vector control were purchased from Sigma Mission® RNAi (Sigma).

Techniques: Expressing, shRNA, Infection, Transduction, Western Blot, Negative Control, Plasmid Preparation, Microarray, Isolation

Journal: Neoplasia (New York, N.Y.)

Article Title: Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer

doi: 10.1016/j.neo.2019.07.001

Figure Lengend Snippet: Chemotherapy downregulates AIB1 expression in TNBC cell lines. (A) HCC1806 and MDA-MB-468 cells were treated as shown (left panel) with DXR, PTX and 5FU. Viable cells were determined by Trypan blue exclusion ( n = 4) (right panel) (B) Total count of HCC1806 cells labeled with Cell Trace Violet dye (left) following chemotherapy treatment ( n = 2) and percent distribution of dividing cells by doubling generations (right). (C) Representative Western blot images for AIB1, E-cadherin, β catenin, and NF-kB from chemotherapy-treated surviving HCC1806 and MDA-MB-468 cells ( n = 2) (D) H&E and AIB1 IHC staining of HCI010 PDX tumor grafts (left) and Western blot images (right) of HCI010 PDX-derived cell lines treated as in A. Graphs are representative of three independent experiments. Technical repeats shown. Mean ± SEM. Scale bar: 200 μm. One-way ANOVA followed by Sidak's (A and C) or Dunnett's (B) multiple comparisons test. * P ≤ .05, ** P ≤ .01, *** P ≤ .001.

Article Snippet: Briefly, 1×10 6 cells/ml densities were fixed, permeabilized, then incubated with 1.2 μg/ml of anti-rabbit human AIB1 (#2126; Cell Signaling Technology, Danvers, MA, USA) primary antibody in suspension for 30–60 minutes.

Techniques: Expressing, Labeling, Western Blot, Immunohistochemistry, Derivative Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer

doi: 10.1016/j.neo.2019.07.001

Figure Lengend Snippet: Phenotype of AIB1 shRNA on BL2-HCC1806 cells in vitro . (A) Cell count per field ( n = 5) (left axis) and percent survival (right axis) of TNBC cell lines following AIB1 shRNA infection and selection relative to their respective control shRNA. (B) Representative Western images for AIB1 in cells from A. (C) Proliferation of serial passaged HCC1806 AIB1 LOW relative to control shRNA in 10%, 1%, 0.1% serum-supplemented RPMI 1640 growth media or serum-free basal media. (D) IC 50 from dose response curves of 72-hours chemotherapy treated HCC1806 AIB1 LOW cells and control shRNA in 1% or 10% serum-supplemented culture conditions. Graphs are representative of three independent experiments. Mean ± SEM. Linear regression (slope coefficient) and Non-linear regression (least-squares) for each cell type. (E) Genomic variant analysis of HCC1806 AIB1 LOW relative to control shRNA (passage 5). Quantification of variant reads (16 variants on 6 chromosomes, n = 2) is shown as a percentage of total reads. One-way ANOVA followed by Dunnett's multiple comparisons test. * P ≤ .05, ** P ≤ .01, *** P ≤ .001.

Article Snippet: Briefly, 1×10 6 cells/ml densities were fixed, permeabilized, then incubated with 1.2 μg/ml of anti-rabbit human AIB1 (#2126; Cell Signaling Technology, Danvers, MA, USA) primary antibody in suspension for 30–60 minutes.

Techniques: shRNA, In Vitro, Cell Counting, Infection, Selection, Control, Western Blot, Variant Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer

doi: 10.1016/j.neo.2019.07.001

Figure Lengend Snippet: AIB1 LOW TNBC cells have reduced tube formation phenotype. (A) Schematic of tube formation assay on Matrigel™. (B) Representative images of 48-hour tube-formation assays showing HCC1806 AIB1 LOW compared to control shRNA cells and (top) and network mask (bottom) (C) Bar graphs showing average of tube network length per well ( n = 6). (D) AIB1 mRNA expression of cells from B. (E) Representative micrographs of 48-hours tube-formation assay of HCC1806 cells that survived chemotherapy vs DMSO control (F) Representative micrographs of tube-formation assays showing AIB1 LOW compared to control shRNA MDA-MB-157 cells at indicated time points. (G) Average tube network length measured per image ( n = 6) of tube-formation from F. Graphs are representative of three independent experiments. Mean ± SEM. Scale bar = 1 mm. Two-tailed t-test. * P ≤ .05, ** P ≤ .01, *** P ≤ .001.

Article Snippet: Briefly, 1×10 6 cells/ml densities were fixed, permeabilized, then incubated with 1.2 μg/ml of anti-rabbit human AIB1 (#2126; Cell Signaling Technology, Danvers, MA, USA) primary antibody in suspension for 30–60 minutes.

Techniques: Tube Formation Assay, Control, shRNA, Expressing, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer

doi: 10.1016/j.neo.2019.07.001

Figure Lengend Snippet: Gene expression patterns of HCC1806 AIB1 LOW cells with reduced tube-formation phenotype. (A) Heatmap showing differentially expressed genes in HCC1806 AIB1 LOW cells relative to control shRNA from tube formation assays. Bold, overlapping genes with 2D cultures. Arrowheads, RT-qPCR validation performed. Gene enrichment plots showing (B) down-regulated and (C) up-regulated pathways in HCC1806 AIB1 LOW cells relative to control shRNA ( n = 2). Gene expression fold change and NES FDR as indicated.

Article Snippet: Briefly, 1×10 6 cells/ml densities were fixed, permeabilized, then incubated with 1.2 μg/ml of anti-rabbit human AIB1 (#2126; Cell Signaling Technology, Danvers, MA, USA) primary antibody in suspension for 30–60 minutes.

Techniques: Gene Expression, Control, shRNA, Quantitative RT-PCR, Biomarker Discovery

Journal: Neoplasia (New York, N.Y.)

Article Title: Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer

doi: 10.1016/j.neo.2019.07.001

Figure Lengend Snippet: Limiting dilution HCC1806 AIB1 LOW cells have reduced growth and metastatic capacity in vivo . (A) Schematic timeline for in vivo limiting dilution analysis. (B) Individual tumor size of HCC1806 AIB1 LOW and control shRNA per LDA group ( n = 10). (C) Representative images of H&E and IHC staining of primary tumor grafts for HCC1806 AIB1 LOW and control shRNA. (D) Volcano plot showing differential gene expression of LDA50-AIB1 LOW xenografts ( n = 2) relative to control shRNA xenografts ( n = 4). (E) PCR gene expression for MMP2 and FN1 in LDA50-AIB1 LOW xenografts ( n = 2) relative to control shRNA xenografts. (F) Number of mice with confirmed pulmonary metastasis by H&E in the LDA50 group. (G) Representative images of H&E, KRT14, and E-cadherin IHC staining in lung tissues for HCC1806 AIB1 LOW and control shRNA. Mean ± SEM. Two-tailed t-test. * P ≤ .05, ** P ≤ .01, *** P ≤ .001.

Article Snippet: Briefly, 1×10 6 cells/ml densities were fixed, permeabilized, then incubated with 1.2 μg/ml of anti-rabbit human AIB1 (#2126; Cell Signaling Technology, Danvers, MA, USA) primary antibody in suspension for 30–60 minutes.

Techniques: In Vivo, Control, shRNA, Immunohistochemistry, Gene Expression, Two Tailed Test