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: Pharmaceutical Biology

Article Title: Methamphetamine leads to the alterations of microRNA profiles in the nucleus accumbens of rats

doi: 10.1080/13880209.2020.1803366

Figure Lengend Snippet: The effects of METH on neuron differentiation by immunofluorescence. The NSE positive cells decreased (A) while GFAP positive cells (B) increased in the striatum, hippocampus, and NAc after METH treatment.

Article Snippet: The primary antibody of NSE (neuron specific enolase, catalog No. AF5169) and GFAP (glial fibrillary acidic protein, catalog No. AF2594) was obtained from the R&D system (Minneapolis, MN, USA).

Techniques: Immunofluorescence

Journal: Cell Death Discovery

Article Title: MUC1 O -glycosylation contributes to anoikis resistance in epithelial cancer cells

doi: 10.1038/cddiscovery.2017.44

Figure Lengend Snippet: Effect of shRNA C1GT suppression on antibody accessibility to cell surface anoikis-initiating molecules. The shRNA C1GT transfected or shRNA control cells from the MUC1- positive F3 cells were incubated with antibodies against E-cadherin, integrin β 1, CD44 or Fas. After washing and application of FITC-conjugated secondary antibody, the cells were analysed by flow cytometry ( a , solid and dotted lines show the peak positions of control and shRNA C1GT transfected cells, respectively. Dotted line shifted to the left or right of the solid line reveals reduction or increase of molecule expression). Mean florescence intensity of the antibody binding are shown in b . Cells were also lysed and analysed by immunoblotting ( c ) for the expression of these molecules with the same antibodies as in a . ** P <0.01, *** P <0.001.

Article Snippet: Antibodies against CD44 (BBA10), integrin β 1 (MAB17782), E-cadherin (MAB1838), Fas (AF2267) and Fas-L (AF126) were from R&D Systems (Abingdon, UK).

Techniques: shRNA, Transfection, Control, Incubation, Flow Cytometry, Expressing, Binding Assay, Western Blot

Journal: Cell Death Discovery

Article Title: MUC1 O -glycosylation contributes to anoikis resistance in epithelial cancer cells

doi: 10.1038/cddiscovery.2017.44

Figure Lengend Snippet: Effect of shRNA C1GT suppression increases antibody accessibility to cell surface anoikis-initiating molecules and enhances Fas-L-induced caspase-8 activity in SW620 cells. shRNA C1GT transfected or shRNA control transfected SW620 cells were incubated with antibodies against E-cadherin, integrin β 1, CD44 or Fas. After application of FITC-conjugated secondary antibody, the cells were analysed by flow cytometry ( a , solid and dotted lines show the peak positions of control and shRNA C1GT transfected cells, respectively. Dotted line shifted to the left or right of the solid line reveals reduction or increase of molecule expression). Mean (±S.D.) florescence intensity of the antibody bindings are shown in b . Cells were also lysed and analysed for the expression of these molecules by immunoblotting with the same antibodies ( c ). In d , the shRNA control and shRNA C1GT transfected SW620 cells were cultured in the presence or absence of 100 ng/ml recombinant Fas-L in suspension and the cell caspase-8 activity was determined after 2 h by Caspase-8-Glo kit. Data are expressed as mean±S.E.M. of triplicate determination of three experiments, *** P <0.001.

Article Snippet: Antibodies against CD44 (BBA10), integrin β 1 (MAB17782), E-cadherin (MAB1838), Fas (AF2267) and Fas-L (AF126) were from R&D Systems (Abingdon, UK).

Techniques: shRNA, Activity Assay, Transfection, Control, Incubation, Flow Cytometry, Expressing, Western Blot, Cell Culture, Recombinant, Suspension

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative bright field (BF) and immunofluorescence images of 5% EtOH treatment (vol/vol, 1 and 3 h) induced neurite retraction, and its regeneration after washing EtOH (Washed, 4 and 20 h) in neuronal PC12 cells. Green: neuron-specific β-III tubulin; Blue: DAPI. Scale bar: 50 µm. B , C Quantification results of mean neurite length per cell and the percentage of cells with neurites. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against neuron-specific β-III tubulin (MAB1195, R&D systems, USA), LC3B (NB100-2220, Novus Biologicals, USA), TOM20 (42406S, Cell Signaling Technology), and Drp1 (8570S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, Control

Journal:

Article Title: The NKG2D-Activating Receptor Mediates Pulmonary Clearance of Pseudomonas aeruginosa

doi: 10.1128/IAI.74.5.2578-2586.2006

Figure Lengend Snippet: NKG2D ligands are induced in the lungs of mice infected with P. aeruginosa. (A) Western blot analysis of RAE-1 in whole lungs of mice exposed to ∼1 × 106 CFU of P. aeruginosa for 0, 8, and 24 h. The blot is representative of results obtained with four to six mice per group. (B to E) NKG2D ligand expression in lung epithelial cells and alveolar macrophages of mice exposed to ∼1 × 106 CFU of P. aeruginosa for 24 h. The accumulation of NKG2D ligands in the lungs of CF1 mice was assessed by immunohistochemistry analysis using paraffin-embedded sections and a goat polyclonal antibody against RAE-1γ. (B) Large airway of PBS-treated mouse. (C) Large airway of P. aeruginosa-infected mouse. (D) Lung parenchyma of PBS-treated mouse. (E) Lung parenchyma of P. aeruginosa-infected mouse. The photomicrographs are representative of five mice per group. Original magnification, ×400.

Article Snippet: The membranes were incubated overnight with anti-RAE-1γ (1-μg/ml dilution; clone AF1136; R&D Systems, Minneapolis, MN).

Techniques: Infection, Western Blot, Expressing, Immunohistochemistry