Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Cells treated with PTC 209 and FMMC 419II cells stably transfected with Bmi1 shRNA plasmid show a decrease in Bmi1 mRNA expression. (B) Purified mRNA from the cells was reverse transcribed into cDNA and then analyzed for Bmi1 mRNA expression with quantitative PCR using TaqMan gene expression assays. The fold difference in expression between control samples and the PTC 209 treated of the Bmi1 shRNA transfected samples was calculated using the average of the Ct (threshold cycle) per group, relative to the expression of the internal control gene GAPDH . (C) FMMC cells were treated with PTC 209 (2 μM and 5 μM) for 24 hours and the expression of Bmi1 protein was detected with western blot. (D) PTC 209 treatment decreased Bmi1 protein expression in FMMC cells. Results are represented as mean ± S.E.M., *P <0.01, ***P <0.005.

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Expressing, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Control, Western Blot

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Untreated (control) FMMC 419II cells. (B) Cells treated with 2 μM PTC 209. (C) Cells treated with 5 μM PTC 209. (D) Results of PTC 209 treatment shown as bar graphs. There is a G0/G1 cell cycle arrest in FMMC 419II cells treated with PTC 209 when compared to untreated cells. Similarly, FMMC 419II cells that have been transfected with a Bmi1 shRNA show a G1 arrest. (E) Bar graphs of cell cycle profiles for FMMC 419II cells from control (F) , colony 2 (G) , colony 4 (H) , and colony 5 (I) . Cells stained with PI/RNAse staining buffer were run on a FACSAria flow cytometer and cell cycle progression was analyzed and quantified (D, E) using FlowJo.

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Control, Transfection, shRNA, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: Flow cytometry analysis of CD49f and CD24 expression for FMCC 419II cells treated with 2 μM PTC 209 versus Bmi1 shRNA transfected colonies 4 and 5 were carried out (A) , and the fluorescent intensities were quantitated (B) .

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Flow Cytometry, Expressing, shRNA, Transfection

Journal: Frontiers in Oncology

Article Title: HER3 Differentiates Basal From Claudin Type Triple Negative Breast Cancer and Contributes to Drug and Microenvironmental Induced Resistance

doi: 10.3389/fonc.2020.554704

Figure Lengend Snippet: Differential HER3 response to AKT and PI3K inhibitors in Basal and Claudin type TNBC. (A) Biochemical assessment of the effect of AKT (GDC0068) and PI3K (GDC0077) inhibition in a panel of Basal and Claudin type Triple Negative Breast Cancer (TNBC) showing expression and upregulation of total and phosphorylated HER3 and downstream signaling in the Basal type TNBC cells (BT-20, HCC70, and MDA-MB-468). Cells were treated for 48h with the respective kinase inhibitors at 1uM (B) HER3 quantification in the TNBC cell lines tested in A and their corresponding mutational status of PTEN, PI3K, KRAS, and BRAF (C) . (D) Biochemical signalling analysis in response to NRG- 1α and -1β in MDA-MB-468 showing increased signalling with NRG- 1β isoform.

Article Snippet: Cells were seeded into 96-well plates followed by treatment with individual kinase inhibitors GDC0068 (Selleckchem), GDC0077, Gefitinib (Selleckchem), Neratinib (Selleckchem), Lapatinib (LC Laboratories), Sapitinib (FisherScientific), and the HER3 Ab (R&D Systems), in the presence or absence of NRG-1α or -1β.

Techniques: Inhibition, Expressing

Journal: Frontiers in Oncology

Article Title: HER3 Differentiates Basal From Claudin Type Triple Negative Breast Cancer and Contributes to Drug and Microenvironmental Induced Resistance

doi: 10.3389/fonc.2020.554704

Figure Lengend Snippet: Neuregulin but not Hepatocyte Growth Factor decreases sensitivity to kinase inhibitors in Basal type Triple Negative Breast Cancer (TNBC). (A) Viability assay assessing the effect of Neuregulin-1β (50 ng/ml) in the presence of the small molecule inhibitors GDC0068 (1uM) and GDC0077 (1uM) after 96h treatment in a panel of TNBC cell lines in BT-20, HCC-70, MDA-MB-468, BT-549, and MDA-MB-231 and (B) Dose dependent effects of NRG- 1α and -1β isoforms [Low Dose (LD) 5 ng/ml, Medium Dose (MD) 50 ng/ml, and high dose (HD) 100 ng/ml] in the presence or absence of GDC0068 (1uM) or GDC0077 (1uM) for 96h in HCC-70, MDA-MB-468, and MDA-MB-231 TNBC. (C) Effect of HGF treatment (50 ng/ml) after 96h in the presence or absence of GDC0068/GDC0077 at 1uM. Drug response graphs and Heat map graphs show CellTiter-Glo luminescence viability measurements at the end of the experiments compared to untreated control. Experiments were performed in triplicate. Data are means ± SD]. Drug response graphs were analyzed using two-way analysis of variance (ANOVA)/Tukey’s multiple comparison test, *<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001].

Article Snippet: Cells were seeded into 96-well plates followed by treatment with individual kinase inhibitors GDC0068 (Selleckchem), GDC0077, Gefitinib (Selleckchem), Neratinib (Selleckchem), Lapatinib (LC Laboratories), Sapitinib (FisherScientific), and the HER3 Ab (R&D Systems), in the presence or absence of NRG-1α or -1β.

Techniques: Viability Assay, Control, Comparison

Journal: Frontiers in Oncology

Article Title: HER3 Differentiates Basal From Claudin Type Triple Negative Breast Cancer and Contributes to Drug and Microenvironmental Induced Resistance

doi: 10.3389/fonc.2020.554704

Figure Lengend Snippet: Gefitinib in combination with AKT/PI3K inhibitors sensitizes Basal type Triple Negative Breast Cancer (TNBC). (A) HCC-70, MDA-MB-468, and MDA-MB-231 TNBC cells treated in the presence of NRG-1β (50 ng/ml) does not significantly decrease sensitivity to gefitinib (200 nM) after 96 h. (B) Viability assay of TNBC cells as in (A) treated with the small molecule inhibitors GDC0068 (1uM), GDC0077 (1uM), gefitinib (200 nM), or a combination of the drugs +/- NRG1β (50 ng/ml) for 96h. In the HCC-70 and MDA-MB-468 cell lines, combination therapy demonstrated decreased viability compared to the MDA-MB-231 cell line which showed no significant difference in response, regardless of treatment (C) Biochemical analysis of the p/EGFR and p/HER3 showing decreased but not abolished phosphorylated proteins indicating a potent and sustained signaling in presence of NRG1β in the Basal type TNBC cells, HCC-70, and MDA-MB-231; Cells were treated for 48h at the drug concentrations shown in (B) . The Claudin type MDA-MB-231 cell line showed no HER3 phosphorylation. Drug response graphs show CellTiter-Glo luminescence viability measurements at the end of the experiments compared to untreated control and analyzed using two-way analysis of variance (ANOVA)/Tukey’s multiple comparison test, *<0.05, ***P<0.001]. Experiments were performed in triplicate. Data are means ± SD].

Article Snippet: Cells were seeded into 96-well plates followed by treatment with individual kinase inhibitors GDC0068 (Selleckchem), GDC0077, Gefitinib (Selleckchem), Neratinib (Selleckchem), Lapatinib (LC Laboratories), Sapitinib (FisherScientific), and the HER3 Ab (R&D Systems), in the presence or absence of NRG-1α or -1β.

Techniques: Viability Assay, Phospho-proteomics, Control, Comparison

Journal: Frontiers in Oncology

Article Title: HER3 Differentiates Basal From Claudin Type Triple Negative Breast Cancer and Contributes to Drug and Microenvironmental Induced Resistance

doi: 10.3389/fonc.2020.554704

Figure Lengend Snippet: Pan HER targeting sensitizes Basal type but not Claudin type Triple Negative Breast Cancer (TNBC) cells to AKT and PI3K inhibition. (A) Biochemical assessment of downstream signaling in the PI3K/AKT signaling pathway after combination therapy with neratinib (300 nM) and GDC0068 (1 uM) or GDC0077 (1 uM) after 48 h, showing deceased signaling in the Basal TNBC cell lines but not in the Claudin type TNBC. (B) Viability assay of TNBC cells treated with the small molecule inhibitors GDC0068, GDC0077, neratinib, or a combination of the drugs for 96h [drug treatments as in (A) ]. In the BT-20, HCC-70, and MDA-MB-468 cell lines, combination therapy demonstrated decreased viability compared to the MDA-MB-231and BT-549 cell line which showed no added benefit of combination therapy. Drug response graphs show CellTiter-Glo luminescence viability measurements at the end of the experiments compared to untreated control and analyzed using two-way analysis of variance (ANOVA)/Tukey’s multiple comparison test, *<0.05, **P < 0.01, ***P<0.001]. Experiments were performed in triplicate. Data are means ± SD].

Article Snippet: Cells were seeded into 96-well plates followed by treatment with individual kinase inhibitors GDC0068 (Selleckchem), GDC0077, Gefitinib (Selleckchem), Neratinib (Selleckchem), Lapatinib (LC Laboratories), Sapitinib (FisherScientific), and the HER3 Ab (R&D Systems), in the presence or absence of NRG-1α or -1β.

Techniques: Inhibition, Viability Assay, Control, Comparison

Journal: Clinical Science (London, England : 1979)

Article Title: New insights into the pathophysiology and therapeutic targets of asthma and comorbid chronic rhinosinusitis with or without nasal polyposis

doi: 10.1042/CS20190281

Figure Lengend Snippet: Targeting immune mechanisms and therapeutic options in T2 and non-T2 inflammation

Article Snippet: , Reactive aldehyde species (RASP) , RASP inhibitor (small molecule) , ADX-629 , Aldeyra , Asthma , 2.

Techniques: Binding Assay, Recombinant, Variant Assay

Journal: Clinical Science (London, England : 1979)

Article Title: New insights into the pathophysiology and therapeutic targets of asthma and comorbid chronic rhinosinusitis with or without nasal polyposis

doi: 10.1042/CS20190281

Figure Lengend Snippet: Most of the current monoclonal antibodies and inhibitors target type 2 inflammation (registered biologics in blue letters). Anti – IgE therapy with omalizumab and ligelizumab is already very effective and safe approach to treat patients with difficult asthma. Similarly, anti-IL-5 monoclonal antibodies, particularly mepolizumab, represent an effective therapy of eosinophilic inflammation. Type 2 inflammation can be downregulated also by dupilumab targeting common subunit shared by IL-4 and IL-13 receptors. Recently, tepelezumab directed against thymic stromal lymphopoietin (TSLP) represents another perspective monoclonal antibody modulating type 2 inflammation with another drugs against alarmins IL-33 and IL-25 in clinical studies. Also the inhibitors of CRTH2 (receptor for PDG2) such as fevipiprant are in clinical studies. In non-type 2 inflammation, no biologics are registered for the therapy of asthma with monoclonal antibodies inhibiting IL-17/IL-23 pathway without sufficient clinical effects, so far. In the effector phases of allergic inflammation, antihistamines and leukotrienes are used for decades with emerging inhibitors of tryptase or reactive aldehyde species (RASP) and monoclonal antibodies blocking pro-inflammatory cytokines IL-6, IL-1 and IL-31 in clinical studies. In the case of anti-TNF therapy, the risk potential adverse effects prevail over benefits in asthma therapy. Finally, chemokine receptor inhibitors may represent another tool to block recruitment of immune cells in allergic inflammation. Created with BioRender Sofware (Ref.n.AB25BY6SZW).

Article Snippet: , Reactive aldehyde species (RASP) , RASP inhibitor (small molecule) , ADX-629 , Aldeyra , Asthma , 2.

Techniques: Blocking Assay