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

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: HER3 activation is a common feedback mechanism of Axl inhibitors, and MPCD84111 blocks phosphorylation of HER3 in contrast to BMS777607. (A) Inhibition of Axl phosphorylation was determined 1 hour posttreatment with BMS777607, MPCD84111, and R428 by p-Tyr–Axl ELISA in NIH/3T3-Axl cells. IC50 values were calculated by four-parameter log curve fit. Axl kinase activity was inhibited in a dose-dependent manner, with an IC50 value of 0.006 μM for BMS777607, 0.027 μM for MPCD84111, and 0.043 μM for R428. (B) Axl Inhibitors induce HER3 expression. Western blot analysis of MDA-MB231 cells treated with 10 μM BMS777607, MPCD84111, and R428 for 24 hours. BMS777607 and R428 caused an increase in pHER3 Y1289 after 24 hours of treatment in contrast to MPCD84111. Treatment with all three Axl inhibitors leads to a six- to 7.5-fold up-regulation of HER3 protein levels. The diagrams show the densitometric analysis of Western blots for pHER3 Y1289 and HER3. Mean values and SEM are shown (n = 3). (C) MPCD84111 blocks phosphorylation of HER3 in contrast to BMS777607. Western blot analysis of MDA-MB231 cells treated with 1 μM BMS777607 or MPCD84111 up to 48 hours is shown. BMS777607 caused an increase in pHER3 Y1289 after 6 hours of treatment in contrast to MPCD84111. Both inhibitors induced a significant increase in protein expression of HER3 after 16 hours of treatment. (D) The kinase selectivity profile against a panel of 36 human kinases proves HER2 as a direct target of MPCD84111. The selectivity profiling was performed in triplicate at a compound concentration of 10 μM. The plots indicate the percentages of inhibition for each individual kinase.

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Western Blot, Concentration Assay

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

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: Inhibition of the Axl RTK leads to up-regulation of HER3 phosphorylation. (A) Inhibition of Axl phosphorylation was determined 1 hour posttreatment with BMS777607 by p-Tyr–Axl ELISA in NIH/3T3-Axl cells. IC50 values were calculated by four-parameter log curve fit. (B) Axl inhibition led to an up-regulation of HER3 phosphorylation as determined by the Human Phospho-RTK Array Kit. MDA-MB231 cells were incubated with 1 μM BMS777607 for 24 hours or Axl-specific siRNA for 48 hours. The capture antibodies have been spotted in duplicates. The coordinates in the membrane for EGFR, HER2, Met, HER3, HER4, InR, IGF1R, and Axl are highlighted by rectangles. The corresponding antibody names are labeled at the bottom of the three displayed membranes from left to the right. (C) Validation of HER3 phosphorylation by HER3 immunoprecipitation with a HER3-specific antibody (Millipore, No. 05-390) and the Western blot for p-Tyr. The site-specific pHER3 Y1289 antibody displayed the same up-regulation of HER3 phosphorylation in Western blot experiments after treatment of MDA-MB231 cells with Axl-specific siRNA for 48 hours. Tubulin served as loading control. (D) Validation of HER3 phosphorylation by Western blot analysis of MDA-MB231 cells treated with Axl-specific siRNA for 48 hours or with 1 or 10 μM BMS777607 for 24 hours. The depletion of Axl kinase by siRNA was confirmed by anti-Axl Western blot analysis. Independent of the conditions, an increase of pHER3 Y1289 levels was evident in contrast to control treatments. Tubulin served as loading control. (E) Met-specific knockdown displayed no effect on pHER3 Y1289 levels compared to control siRNA treatment. The phosphorylation of HER3 Y1289 was induced by 10 μM BMS777607 treatment for 24 hours independent of Met expression. The depletion of Met kinase from the cells was confirmed by Western blot analysis performed on cell lysates harvested 48 hours after Met-specific siRNA transfection. Tubulin served as loading control.

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

Techniques: Inhibition, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Membrane, Labeling, Biomarker Discovery, Immunoprecipitation, Western Blot, Control, Knockdown, Expressing, Transfection

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

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: Inhibition of the Axl RTK activates HER3 transcription and phosphorylation. HER3 correlates with consumption of NRG1. (A) Quantification of HER3 mRNA induction in MDA-MB231 cells. Cells were treated with 10 μM BMS77607 for 1 hour up to 48 hours. A three- to six-fold increase of HER3 mRNA was evident between 16 to 48 hours posttreatment. Mean values and SEM are shown (n = 3). (B) Quantification of NRG1 mRNA induction with primer set 1 detecting all isoforms of NRG1 except isoforms No. 2 and No. 14. Cells were treated with 10 μM BMS777607 for 1 hour up to 48 hours. No significant increase of NRG1 mRNA levels was detected. Mean values and SEM are shown (n = 3). (C) Quantification of NRG1 mRNA induction with primer set 2 detecting isoforms No. 2, No. 15, and No. 17 of NRG1. Cells were treated with 10 μM BMS777607 for 1 hour up to 48 hours. No significant increase of NRG1 mRNA levels was detected. Mean values and SEM are shown (n = 3). (D) Quantification of NRG1 protein levels in conditioned medium was assayed using NRG1-ELISA. Cells were treated with 10 μM BMS777607 for 1 to 48 hours. A time-dependent consumption of NRG1 was measured. Mean values and SEM are shown (n = 3). (E) Western blot analysis of MDA-MB231 cells. Cells were treated with 10 μM BMS777607 for 1 to 48 hours. A significant increase of the pHER3 Y1289 levels was evident in MDA-MB231 after 6 hours of treatment. The up-regulation of HER3 protein expression was evident 16 hours posttreatment. BMS777607 treatment continuously suppressed the phosphorylation of AKT S473. Tubulin served as loading control. The diagrams show the densitometric analysis of Western blots for pHER3 Y1289 and HER3. Mean values and SEM are shown (n = 3).

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

Techniques: Inhibition, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

doi: 10.1371/journal.pone.0118713

Figure Lengend Snippet:

Article Snippet: Phosphorylated AKT (Cat# AF887, R & D Systems, Minneapolis, MN) , 5% BSA in TTBS , 1:400 , goat anti-rabbit.

Techniques: Blocking Assay, Concentration Assay

Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 4. NT regulates miR-21 and miR-155 signaling pathways in colon cancer cells. (A) Luciferase activity of the PTEN and SOCS1 3=UTRs after NT treatment (100 nmol/L) for 24 hours in HCT116 and DLD1 cells (untreated, as-miR-NC–treated, as-miR-21–treated, or as-miR-155–treated). (B) PTEN and SOCS1 mRNA levels, assessed by real-time PCR, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells (untreated, as-miR-NC– treated, as-miR-21–treated, or as-miR-155–treated). (C) PTEN, SOCS1, and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (D) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), and/or as-miR-155 (100 nmol/L) for 24 hours and treated with NT (100 nmol/L) for 24 hours.

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Protein-Protein interactions, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 5. NT activates AKT through suppression of PPP2CA by direct interaction with miR-155. (A) miR-155 binding sites in 3=UTRs of PPP2CA predicted by Lever algorithm analysis. (B) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells transfected with as-miR-155 (100 nmol/L) or as-miR-155 (100 nmol/L) for 24 hours. (C) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells treated with NT (100 nmol/L). (D) PPP2CA and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (E) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with miR-155 (100 nmol/L) or si-PPP2CA (100 nmol/L) for 24 hours. (F) NF-B/p65 activity, assessed by enzyme-linked immunosorbent assay, in HCT cells transfected with NT (100 nmol/L) and as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), as-miR-155 (100 nmol/L), or with 10 nmol/L of an AKT pharmacologic inhibitor (MK-2206).

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: The American Journal of Pathology

Article Title: Lung-Residing Metastatic and Dormant Neuroblastoma Cells

doi: 10.1016/j.ajpath.2011.03.020

Figure Lengend Snippet: The Malignancy Phenotype of MetNB and DisNB Cell Variants

Article Snippet: For Western blot analysis: anti-extracellular signal-regulated kinase2 (C-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-phosphorylated-ERK1/2 (R&D Systems) at 1:1000; anti-phosphorylated AKT (S473, R&D Systems) at 1:2000; anti AKT1 (B-1, Santa Cruz Biotechnology at 1:1000; anti-paracingulin (CGNL1, Zymed Laboratories Inc., San Francisco, CA) at 1:500; anti-matrix metalloproteinase (MMP)-2 and anti-MM9 at 1:500 μg/mL (EMD Chemicals, Inc., San Diego, CA).

Techniques: Migration