Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: (A) 8MOP interacts with three peptide regions within the ErbB2 catalytic kinase domain. Qualitative peptide identifications within the ErbB2 catalytic kinase domain following LC-MS/MS analysis of a streptavidin pull-down of biotinylated-8MOP bait (see ). The transmembrane domain is indicated (red diamond) and the five C-terminus tyrosine autophosphorylation sites are indicated (p). (B) Non-reducing Western blot analysis of the interaction of 8MOP with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or with vehicle (0.01% DMSO) alone served as control for 48 hr and then exposed to UV irradiation (2J) prior to Western blot analysis. The image on the left shows the Western blot for ErbB2 (red). The image on the right shows the same membrane directly scanned for the presence of 800dye-8MOP (green), which overlays the ErbB2 signal. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Irradiation, Membrane

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: The growth and viability of BT474 and SKBR3 cells (top bar graphs) after being subjected to the indicated treatment conditions. The combination of PUVA plus neratinib: P <0.0005 (BT474 and SKBR3 cells). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) Western blot analysis showing steady-state ErbB2, ErbB3, and phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated according to the indicated treatment conditions. Vehicle alone (0.01% DMSO) served as a control. Steady-state actin protein levels served as a control for equal loading of protein. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: Top bar graph shows the results of the growth assays performed in T47D and stably transfected T47D cell line. T47D cells expressing p85 ErbB2 were pretreated with 5 µM lapatinib or 5 µM 8MOP for 4 hr followed by irradiation in a UV Stratalinker 1800 (Statagene). Cells transfected with empty vector (T47D/Vector), and those treated with vehicle alone (0.01% DMSO) served as controls. The effects of the treatments on cell growth and viability are shown in the bar graph. P<0.0071 (8MOP + UVA irradiation). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Steady-state phospho-p85 ErbB2 protein levels (dotted arrow) and phospho-p185 ErbB2 (solid arrow) are shown by Western blot. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Stable Transfection, Transfection, Expressing, Irradiation, Plasmid Preparation, Western Blot, Control

Journal: BMC Cancer

Article Title: uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R

doi: 10.1186/s12885-016-2663-9

Figure Lengend Snippet: Co-overexpression of uPAS components and tumour-promoting proteins in TNBC samples and cell lines. a Immunohistochemical analysis of tumour samples showing positive expression and localisation of the proteins of interest: uPAR, uPA, PAI-1, IGF1R, IR and c-Met, bar: 50 μm and b Protein expressions of uPAR, uPA, PAI-1, IGF1R, IR and c-Met in the TNBC cohort ( n = 174). c Immunoblottings of uPAR, uPA (supernatant), PAI-1, IGF1R, (phospho) c-Met, HER2, ER, PR in two TNBC cell lines: BT549 and MDA-MB-231 and in the breast cancer cell lines: BT474, MCF7, MDA-MB-361, SKBR3 and T47D. Tubulin was used as loading control

Article Snippet: The following human breast cancer cells lines MDA-MB-361 (HTB-27), SKBR3 (HTB-30), T47D (HTB-133) were acquired from American Type Culture Collection (ATCC) and MCF7 (ACC115) cells from German Collection of Microorganisms and Cell Cultures, DSMZ).

Techniques: Over Expression, Immunohistochemical staining, Expressing, Control

Journal: PLoS ONE

Article Title: 3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

doi: 10.1371/journal.pone.0054657

Figure Lengend Snippet: ( A ) SKBR3 and MDA-MB-468 cells were treated with DIM (15 µM) and Herceptin (0.75 µg/ml), either alone or combination for 72 hours. DIM in combination with Herceptin significantly inhibited cell proliferation, as measured by MTT assay. ( B ) SKBR3 and MDA-MB-468 cells were treated with 15 µM DIM, 0.75 µg/ml Herceptin and combination. Assay for anchorage-dependent clonogenicity was done as described under materials and methods. ( C ) The bar graphs at the bottom represent quantification of results presented on the top in each case. *, P <0.05; **, P <0.01 relative to control. C, Control; D, DIM; H, Herceptin.

Article Snippet: Breast cancer cell lines, SKBR3 ( HER-2/neu -expressing) and MDA-MB-468 ( HER-2/neu negative) were obtained from ATCC (Manassas, VA).

Techniques: MTT Assay, Control

Journal: PLoS ONE

Article Title: 3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

doi: 10.1371/journal.pone.0054657

Figure Lengend Snippet: ( A ) Induction of apoptosis in SKBR3 and MDA-MB-468 cells treated with 15 µM of DIM, 0.75 µg/ml of Herceptin, and their combination. Fold changes in DIM and Herceptin-treated SKBR3 and MDA-MB-468 cells relative to untreated cells are shown. ( B ) PARP cleavage assay showed that combination treatment with DIM and Herceptin induced significantly greater apoptosis. The p values (**, P <0.01) represent comparisons between cells treated by either of the drugs and their combinations by using the paired t test. C, Control; D, DIM (15 µM/L DIM); H, Herceptin (0.75 µg/ml).

Article Snippet: Breast cancer cell lines, SKBR3 ( HER-2/neu -expressing) and MDA-MB-468 ( HER-2/neu negative) were obtained from ATCC (Manassas, VA).

Techniques: Cleavage Assay, Control

Journal: PLoS ONE

Article Title: 3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

doi: 10.1371/journal.pone.0054657

Figure Lengend Snippet: Expression of FoxM1, Akt, pAkt and NF-κB p65 , and β-actin in SKBR3 and MDA-MB-468 cell lines treated with 15 µM DIM, 0.75 µg/ml Herceptin or the combination for 72 hours. The bar graphs at the bottom in each case are based on quantitative values of bands of interest normalized to β-actin. *, P <0.05 and **, P <0.01 relative to control.

Article Snippet: Breast cancer cell lines, SKBR3 ( HER-2/neu -expressing) and MDA-MB-468 ( HER-2/neu negative) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: 3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

doi: 10.1371/journal.pone.0054657

Figure Lengend Snippet: ( A ) Comparative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells by real-time miRNA RT-PCR. ( B and C ) Relative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells treated with 15 µM DIM, 0.75 µg/ml Herceptin or their combinations for 24 h. ( D ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on cell proliferation of SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as measured by MTT assay. ( E ) Efficacy of anti-miR-200s transfections as well the effect of DIM plus Herceptin (15 µM DIM and 0.75 µg/ml Herceptin for 72 hours) on expression levels of anti-miR-200s (miR-200a/miR-200b/miR-200c) was evaluated by real time RT-PCR. ( F ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on expression of FoxM1 and pAkt in SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as determined by western blotting. The numbers represent percent of corresponding control normalized to β-actin levels. Her, Herceptin; anti-miR-200s, anti-miR-200a+anti-miR-200b+anti-miR-200c. *, P <0.05 and **, P <0.01 relative to control.

Article Snippet: Breast cancer cell lines, SKBR3 ( HER-2/neu -expressing) and MDA-MB-468 ( HER-2/neu negative) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, miRNA RT, MTT Assay, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: PLoS ONE

Article Title: 3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

doi: 10.1371/journal.pone.0054657

Figure Lengend Snippet: Expression of FoxM1 , Akt, pAkt and NF-κB p65 in SKBR3 and MDA-MB-468 cells after transfection with pre-miRNAs (pre-miR-200a+pre-miR-200b+pre-miR-200c) and FoxM1 siRNA followed by 15 µM of DIM and 0.75 µg/ml of Herceptin for 48 h. D, DIM; H, Herceptin. The numbers represent percent of corresponding control normalized to β-actin levels.

Article Snippet: Breast cancer cell lines, SKBR3 ( HER-2/neu -expressing) and MDA-MB-468 ( HER-2/neu negative) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Control

Journal: Cell chemical biology

Article Title: The Advantages of Targeted Protein Degradation over Inhibition: a RTK Case Study

doi: 10.1016/j.chembiol.2017.09.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies EGFR SantaCruz 1005 FLAG Sigma F1804 Tubulin Sigma T9026 HER2 Cell Signaling 2165S p-EGFR (Y1068) Abcam ab40815 p-HER2 (Y1221/1222) Cell Signaling 2243S p-AKT (T308) Cell Signaling 2965S p-ERK 1/2 (T202/204) Cell Signaling 4370 HER3 Cell Signaling 12708 pHER3 (Y1197) Cell Signaling 4561 pHER3 (Y1289) Cell Signaling 4791 pGSK-3b (S9) Cell Signaling 9331 c-Met Cell Signaling 8198 c-Met Cell Signaling 3127 p-Met (Y1245/1235) Cell Signaling 3126 p-AKT (S473) Cell Signaling 4060 Ubiquitin (P4D1) Cell Signaling 3936 p230 Trans Golgi BD Biosciences 611281 EEA1 BD Biosciences 610456 Clathrin Heavy Chain SantaCruz sc-12734 Alexa Fluor-546 conjugated anti-mouse ThermoFisher A-21143 Alexa Fluor-488 conjugated anti-rabbit ThermoFisher A-11008 HRP linked Mouse IgG GE Life Sciences NA931 HRP Linked Rabbit IgG GE Life Sciences NA934 Chemicals, Peptides, and Recombinant Proteins Cycloheximide Sigma C104450 MLN4924 Sigma 5.05477 PR-619 LifeSensors SI9619 EZ-link Sulfo-NHS-SS-Biotin Thermo 21331 Pierce NeutrAvidin Agarose beads Thermo 29200 Agarose-TUBE 1 LifeSensors UM401 Protein A-Sepharose 4B, Fast Flow beads Sigma P9424 Recombinant Human HGF Protein R&D Systems 294-HG-250 Critical Commercial Assays CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) Promega G5421 Experimental Models: Cell Lines OVCAR8 Joyce Liu, Dana Farber MDA-MB-231 ATCC HTB-26 HeLa ATCC CCL-2 HCC827 ATCC CRL-2868 H3255 Katerina Politi, Yale H1975 ATCC CRL-5908 SKBr3 ATCC HTB-30 GTL16 F. Maina, Developmental Biology Institute of Marseille-Luminy Hs746T ATCC HTB-135 Oligonucleotides Clathrin Heavy Chain siRNA SantaCruz sc-35067 Software and Algorithms Image Lab 6.0 Biorad N/A Graphpad Prism N/A Open in a separate window Key Resources Table

Techniques: Ubiquitin Proteomics, Recombinant, Proliferation Assay, Software