Journal: Oncogene

Article Title: Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells.

doi: 10.1038/sj.onc.1209113

Figure Lengend Snippet: Figure 1 PKC induces neurofibromin phosphorylation in neural cells. (a) and (b) TPA (100 nM) or (100 ng/ml) EGF induced significant increases in [32P]-orthophosphate incorporation in neurofibromin, in C62B rat astrocytoma cells and primary neurons from 6-day chick embryo telencephalon (E6) grown in culture for 2 (C2) or 7 days (C7). Cell lysates were immunoprecipitated with neurofibromin antibodies, and immunoprecipitates were analyzed by SDS–PAGE and dried gel autoradiography. Panel a contains a typical autoradiogram of immunoprecipitated neurofibromin and panel b is the image of Coommassie stain of the same SDS gel (7% polyacrylamide) prior to drying. (c) The magnitude of response appeared developmentally regulated and was higher in immature neurons E6C2 than in differentiated E6C7 neurons (autoradio- graphy of 4–12% gradient gels). The response was completely abolished by the PKC inhibitor chelerythrine chloride (CC, 5 mM), but not by the peptide PKA inhibitor pPKAi (10 ng/ml). (d) Statistical evaluation of these counts confirmed the autoradio- graphy data. In all experiments corresponding gels areas were excised and [32P] incorporation was quantitated by scintillation counting; bracketed bars in graph represent the mean7s.e. values from 15–20 samples from five to eight different experiments and *Po0.001 over time 0.

Article Snippet: PKC inhibitors Gö6976 and rottlerin were from Calbiochem, chelerythrine chloride from and PKA-specific inhibitor pPKAi were from Alexis and Santa Cruz, respectively.

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Autoradiography, Staining, SDS-Gel

Journal: Oncogene

Article Title: Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells.

doi: 10.1038/sj.onc.1209113

Figure Lengend Snippet: Figure 3 PKC activation-dependent association of neurofibromin with actin. (a) Amounts of neurofibromin co-immunoprecipitated with anti-b-actin monoclonal mouse IgG1 (1 mg of IgG1 per 1 mg of total cell lysate) in immature E6 cerebral neurons at day 2 in culture (C2) were significantly enhanced with time of exposure to TPA, as seen by Western blotting with anti-neurofibromin antibodies (upper panel) after SDS–PAGE analysis on 7–16% gels. (b) Quantitation by densitometry of neurofibromin co-immunoprecipitated with actin antibodies in neurons, expressed as ratio of neurofibromin densities to actin, and statistical analysis. (c) Similar increases in association of neurofibromin with actin upon treatment with TPA in SH-SY5Y neuroblastoma cells were abolished by pretreatment with the PKC inhibitor chelerythrine chloride (CC, 5 mM) (SDS–PAGE analysis on 4–12% gels). (d) Quantitation by densitometry of neurofibromin co-immunoprecipitated with actin antibodies in SH-SY5Y. (e) EGF stimulation also increased the association of neurofibromin with actin in C62B cells in a PKC-dependent manner (CC but not pPKAi-dependent), in a more potent manner than TPA (SDS–PAGE analysis also on 4–12% gels). Left arrows point to the 215 kDa prestained marker in (a, c), and left line in (e) indicates the place of the same prestained marker. Bars in (b, d, f) represent the mean 7s.e. values of 3–8 samples from five different experiments, *Po0.05, and **Po0.02.

Article Snippet: PKC inhibitors Gö6976 and rottlerin were from Calbiochem, chelerythrine chloride from and PKA-specific inhibitor pPKAi were from Alexis and Santa Cruz, respectively.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, SDS Page, Quantitation Assay, Marker

Journal: Molecules

Article Title: Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

doi: 10.3390/molecules25010224

Figure Lengend Snippet: Dose–response curves of chelerythrine chloride treatments in NCI-H1703, SK-LU-1, and human lung cancer stem cells (HLCSC). Representative dose–response curves show the kinetic response of ( A ) NCI-H1703, ( B ) SK-LU-1, and ( C ) HLCSC toward chelerythrine chloride in a span of 72 h treatments. Curves show dose–response of NCI-H1703, SK-LU-1, and HLCSC toward chelerythrine chloride after ( D ) 24, ( E ) 48, and ( F ) 72 h treatment. Dose–response kinetic curves in ( A – C ) were plotted from duplicate data obtained in a representative of two independent experiments. Dose–response curves in ( D – F ) were derived from means of CI at specified time-points from two independent experiments.

Article Snippet: Chelerythrine chloride was purchased from Chromadex (Lost Angeles, CA, USA).

Techniques: Derivative Assay

Journal: Molecules

Article Title: Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

doi: 10.3390/molecules25010224

Figure Lengend Snippet: IC 20 , IC 50 , and IC 80 values of chelerythrine chloride.

Article Snippet: Chelerythrine chloride was purchased from Chromadex (Lost Angeles, CA, USA).

Techniques:

Journal: Molecules

Article Title: Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

doi: 10.3390/molecules25010224

Figure Lengend Snippet: Molecular implications of chelerythrine chloride treatment in lung cancer cell lines. NCI-H1703, SK-LU-1, and HLCSC were treated with GSK3i for 24 h and sequentially treated with various concentrations of chelerythrine chloride for 24 h. Subsequently, immunofluorescence stainings of β-catenin were compared among the cell lines. ( A ) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in NCI-H1703. ( B ) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in SK-LU-1. ( C ) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in HLCSC. Scale bars represent 100 μm. ( D ) Representative quantification shows cell number with positive nuclear β-catenin in an independent immunostaining experiment. Error bars are expressed as mean ± SD. Most responsive chelerythrine chloride-treated cell lines—NCI-H1703′s—protein lysates were resolved using Western blotting. ( E ) Representative Western blots show the effect of chelerythrine chloride treatment toward the expression of CSC-related transcription factors, namely β-catenin, MYC, and SOX2. Statistical significance was expressed as *** p < 0.001.

Article Snippet: Chelerythrine chloride was purchased from Chromadex (Lost Angeles, CA, USA).

Techniques: Immunofluorescence, Immunostaining, Western Blot, Expressing

Journal: Molecules

Article Title: Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

doi: 10.3390/molecules25010224

Figure Lengend Snippet: Chelerythrine chloride induces apoptosis and inhibits CSC functions. ( A ) Parallel estimations of cell viability, cytotoxicity and apoptosis were performed after 24 h treatment of chelerythrine chloride in NCI-H1703 at indicated concentrations using ApoTox-Glo triplex assay. ( B ) Representative micrographs show three weeks grown soft agar colonies of NCI-H1703 upon treatment of chelerythrine chloride at indicated concentrations. Scale bars represent 1000 μm. ( C ) Representative micrographs show spheroids’ morphological characteristics after 24 h treatment of chelerythrine chloride at indicated concentrations. Scale bars represent 200 μm. ( D ) Dose–response bar graph show comparison of cytotoxicity of various concentration of chelerythrine chloride between monolayer and spheroid models of NCI-H1703. ( E ) Estimations of migration and invasion of NCI-H1703 after 16 h of chelerythrine chloride treatment at indicated concentrations using real-time cell analyzer (RTCA). ( F ) Effect of corresponding treatment concentrations in ( E ) to cell viability is shown in bar graph. All data were obtained from mean of three independent experiments. Error bars are expressed as mean ± SD. Statistical significance was expressed as *** p < 0.001 ** p < 0.01; * p < 0.05.

Article Snippet: Chelerythrine chloride was purchased from Chromadex (Lost Angeles, CA, USA).

Techniques: Comparison, Concentration Assay, Migration

Journal: Plants

Article Title: The Cultivation of Chelidonium majus L. Increased the Total Alkaloid Content and Cytotoxic Activity Compared with Those of Wild-Grown Plants

doi: 10.3390/plants10091971

Figure Lengend Snippet: List of tentatively identified alkaloids in the ethanol extracts of aerial parts of C. majus.

Article Snippet: The reference substances sanguinarine, chelerythrine, and chelidonine were purchased from Biosynth Carbosynth (Compton, UK), Alfa Aesar Chemicals (Heysham, UK), and Cayman Chemical (Ann Arbor, MI, USA), respectively.

Techniques:

Journal: Plants

Article Title: The Cultivation of Chelidonium majus L. Increased the Total Alkaloid Content and Cytotoxic Activity Compared with Those of Wild-Grown Plants

doi: 10.3390/plants10091971

Figure Lengend Snippet: Content of alkaloids (µg/g of dry material) in the ethanol extracts of aerial parts of wild-grown and cultivated C. majus.

Article Snippet: The reference substances sanguinarine, chelerythrine, and chelidonine were purchased from Biosynth Carbosynth (Compton, UK), Alfa Aesar Chemicals (Heysham, UK), and Cayman Chemical (Ann Arbor, MI, USA), respectively.

Techniques: