Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA activates Src kinase in A549 cells, but reduces the activity of recombinant Src kinase in a cell-free assay. (A) A549 cells were treated with AcoA for 1 to 24 h, lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, ** p < 0.01, *** p < 0.001 in comparison to cells incubated with vehicle for the indicated time. (B) AcoA, ouabain and digoxin reduce Src kinase activity in a cell-free assay. Enzymatic activity of recombinant c-Src kinase (0.1 U/mL) pretreated with AcoA, ouabain, digoxin, doxorubicin, or the positive control staurosporine (all 100 nM) for 30 min, was analyzed by a kinase assay. Data are mean ± SD of triplicates, * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to the vehicle control. (C) Src activation ( p -Src Y416 expression) in the NCI-60 tumor cell lines does not correlate with AcoA cytotoxicity (GI 50 values) in the same cell lines (original data were obtained from NCI database). Red dot indicates A549 cell line. Pearson correlation test. (D) AcoA and ouabain-induced cell toxicity is unaffected by the Src inhibitor PP2. Cell viability of A549 cells pretreated for 4 h with the Src kinase family inhibitor PP2 and incubated for 48 h with AcoA or ouabain (100 nM). XTT assay, mean ± SEM of N = 3.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Activity Assay, Recombinant, Cell-Free Assay, Activation Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Positive Control, Kinase Assay, Control, Expressing, XTT Assay

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA leads to endosomal EGFR arrest and inhibits EGF-induced degradation of EGFR (A) AcoA induces endosomal arrest in EGFR biosensor cells. After 1 h preincubation with monensin (1 µM), AcoA, digoxin, doxorubicin (all 100 nM), or vehicle, cells were stimulated with 100 ng/ml EGF and the formation of green fluorescent vesicles indicating activated and internalized EGFR was monitored microscopically. Representative images are shown. The graphs on the right show the number of fluorescent vesicles/cell at the respective time point. Data are mean ± SEM of N = 3 independent experiments, *** p < 0.001. (B) Quantification of cellular EGFR in A549 cells treated as in (A) using a commercial ELISA In-cell ELISA assay. The amount of EGFR was normalized to cell number assessed by crystal violet staining. Data are mean ± SEM of N = 3 independent experiments, * p < 0.05 vs. control, # p < 0.05 vs. EGF treatment group. (C) At the same conditions, EGFR activation was assessed using ELISA for the Y1173 phosphorylation site of EGFR. EGFR phosphorylation is expressed as the ratio of phosphorylated EGFR to total EGFR. Data are mean ± SEM of N = 3, * p < 0.05 vs. control. (D) Src kinase activation was measured as a downstream target of EGFR. A549 cells treated as in (A) were lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, * p < 0.05 vs. control, ** p < 0.01 vs. control, # p < 0.05 vs. EGF treatment group.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Staining, Control, Activation Assay, Phospho-proteomics

Journal: Cancers

Article Title: Rac1 Signaling: From Intestinal Homeostasis to Colorectal Cancer Metastasis

doi: 10.3390/cancers12030665

Figure Lengend Snippet: Upstream effectors; RhoGAsP, RhoGDIs, GEFs.

Article Snippet: PLCG1, PLC-gamma-1 , Phospholipase C Gamma 1 , Hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,4,5-trisphosphate (IP3) and diacylglycerol , Critical for EGF-induced Rac1 activation in vivo. PLC-g SH3 domain acts as a Rac1 guanine nucleotide exchange in vivo. Cytoskeleton remodeling and cell migration [ ]. , .

Techniques: Activity Assay, Migration, Membrane, Binding Assay, Over Expression, Microarray, Activation Assay, Expressing, Adhesive, Mutagenesis, Genome Wide, Variant Assay, Blocking Assay, In Vivo

Journal: Cancers

Article Title: Rac1 Signaling: From Intestinal Homeostasis to Colorectal Cancer Metastasis

doi: 10.3390/cancers12030665

Figure Lengend Snippet: Interacting partners and functional consequences.

Article Snippet: PLCG1, PLC-gamma-1 , Phospholipase C Gamma 1 , Hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,4,5-trisphosphate (IP3) and diacylglycerol , Critical for EGF-induced Rac1 activation in vivo. PLC-g SH3 domain acts as a Rac1 guanine nucleotide exchange in vivo. Cytoskeleton remodeling and cell migration [ ]. , .

Techniques: Functional Assay, Membrane, Activity Assay, Activation Assay, Mutagenesis, Over Expression, Binding Assay, Migration, Translocation Assay, Expressing, Transduction, Scaffolding, Control, Sequencing, Extraction, Inhibition, Knockdown

Journal: Frontiers in Immunology

Article Title: Tumor Cell-Secreted ISG15 Promotes Tumor Cell Migration and Immune Suppression by Inducing the Macrophage M2-Like Phenotype

doi: 10.3389/fimmu.2020.594775

Figure Lengend Snippet: Interferon-stimulated gene 15 (ISG15)-induced CCL18 secretion by macrophages was dependent on the ISG15 receptor, leukocyte function-associated antigen-1 (LFA-1), and SRC family kinase (SFK) signaling. (A) Immunofluorescence demonstrated that the ISG15 receptor CD11a/CD18, also called LFA-1, was expressed on the membranes of human macrophages. Macrophages were treated with 295nM rISG15 for 24 h. Scale bars, 10 μm. (B) ELISA results showed small molecular inhibitors of LFA-1, A286982, inhibited CCL18 secretion by macrophages. (C) Small molecular inhibitors of LFA-1 and SRC, A286982 and PP2, hindered the ISG15-induced activation of SFK signaling. (D) PP2 reduced the CCL18 secretion by ISG15-treated macrophages in a dose-dependent manner, as determined by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Protein extracts were resolved by 10% SDS–PAGE, transferred to PVDF membranes (Roche), and probed with antibodies directed against human ISG15 (1:1,000; Abnova, catalog no. A155801), SRC (1:1,000; CST, catalog no. 9935T), and β-actin (1:3,000; Abcam, catalog no. ab69512).

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Cells

Article Title: Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction

doi: 10.3390/cells10081871

Figure Lengend Snippet: Mechanism of action of triazinetriones on TrkA. ( a ) Full-length TrkA with a HA-tag (TrkA-HA) fused to the C -terminus was purified through immunoprecipitation using anti-HA agarose beads. Purified TrkA-HA was incubated with DMSO (blue circles and hatched line), ACD855 (5 µM) (black triangles and solid line), or ACD856 (1 µM) (red squares and solid line) for approx. 5 min. Thereafter, ATP was added to yield the indicated concentration. Each data point is the mean ± SEM ( n = 3). The solid lines are the curve-fit using the Michaelis–Menten equation used to calculate the apparent Vmax(app) and km(app), and the dotted lines are the 95% confidence band for each curve fit. ( b – d ) Affinity labeling and streptavidin adsorption of Trka. ( b ) Western blot of streptavidin adsorbed TrkA-HA non-covalent labeled with NHS-biotinylated triazinetrione compound (lane 2) or covalent labeled by UV-crosslinking of 100 µM sulfo-SBED biotinylated compound (lane 3). Detection of immunoreactive band was performed with anti-TrkA antibody. Supernatant loaded to the left (lane 1) was used as positive control for the Western blot. Arrows on the left indicate the migration of molecular weight markers corresponding to 198, 98, and 62 kDa. ( c ) Anti-HA agarose immunoprecipitation of cross-linked or non-crosslinked sulfo-SBED compound (AC27019-SBED) from cell lysate incubated with 100 µM AC27019-SBED. Lane 1, non-UV-crosslinking, and lane 2, UV-crosslinking of sulfo-SBED labeled TrkA-HA, both detected with streptavidin-HRP. Lanes 3 and 4 are loading controls of lanes 1 and 2, respectively, were TrkA-HA was detected by immunoblotting using the anti-TrkA antibody. Both blots were part of the same gel, but the membrane was cut in two pieces, and the proteins were detected by streptavidin-HRP (left panel) or by an anti-TrkA antibody (right panel). ( d ) Streptavidin adsorption of biotinylated compound bound to TrkA-HA. Lane 1, cell lysate used for positive control of immunodetection; lane 3, cell lysate without biotinylated compound was adsorbed to streptavidin-agarose as negative control; lanes 5 and 7, cell lysates containing sulfo-SBED compound UV-crosslinked to TrkA (from two different experiments) were adsorbed to streptavidin-agarose and immunoblotted using anti-TrkA antibody. Lanes 2, 4, and 6 are empty lanes to avoid cross-contamination between lanes.

Article Snippet: Recombinant intracellular domain (ICD) of TrkA (08-186) and single site biotinylated activated (08-486-20N) or non-activated TrkA (08-486-23N) were obtained from Carna Biosciences, Odense, Denmark.

Techniques: Purification, Immunoprecipitation, Incubation, Concentration Assay, Labeling, Adsorption, Western Blot, Positive Control, Migration, Molecular Weight, Membrane, Immunodetection, Negative Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Hepatic activity on the 3D in vitro model stimulated with the combination of NAC 12 mM in the presence of VitD3 100 nM and Glutathione 5 mM and compared to LA 50 µM. ( a ) Cell viability measured by MTT test; ( b ) TNFα measurement by ELISA kit; ( c ) NF-Kb measurement by ELISA kit; ( d ) NAC dosage performed with spectrophotometry technique. Data are expressed as means ± SD (%) of five independent experiments performed in triplicate and normalized to control values (the 0% line). In ( a – c ), * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM. In ( d ), * p < 0.05 vs. control and the bars p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Spectrophotometry, Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Analysis of intracellular markers involved in hepatic homeostasis on a 3D in vitro liver model after 24 h. ( a ) CYP1A2, ( b ) CYP3A4, ( c ) SRC, and ( d ) ERK/MAPK activities measured by ELISA kit. Data are mean ± SD of five independent experiments performed in triplicates and expressed as percentages (%) vs. control values (0% line). * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Analysis of the beneficial effects on CCF-STTG1 cells. ( a ) NAC quantification by spectrophotometry technique and ( b ) cell viability measured by MTT test in a study from 1 h to 6 h; ( c ) ROS production analysis measured through cytochrome C reduction in CCFTG cells after 24 h; ( d ) TNFα and ( e ) IL1- β activities measured by ELISA kit on CCFTG cells after 24 h. Data are mean ± SD of five independent experiments performed in triplicates vs. control values (0% line). * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Analysis of biomarkers in CCF-STTG1 cells. ( a ) pTAU activity and ( b ) APP activity measured by ELISA kit on CCFTG cells after 24 h. Data are mean ± SD of five independent experiments performed in triplicates vs. control values (0% line). * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Effects of the substances on PNS in vitro model after 24 h. ( a ) Cell viability measured by the MTT test; ( b ) TNFα and ( c ) IL1- β activities analysed by ELISA kit; ( d ) p75 activity performed by ELISA kit; ( e ) NAC concentration determined by spectrophotometry technique. In all panels, VitD3 100 nM and Glut 5 mM are also reported. Data are mean ± SD of five independent experiments performed in triplicates vs. control values (0% line). * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay, Concentration Assay, Spectrophotometry, Control

Journal: Foods

Article Title: The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal–Neuronal In Vitro Model

doi: 10.3390/foods13050774

Figure Lengend Snippet: Analysis of Schwann cell activity after 24 h. ( a ) p75; ( b ) MPZ; ( c ) NRG1; ( d ) ERB3 activities analysed by ELISA kit. Data are mean ± SD of five independent experiments performed in triplicates vs. control values (0% line). * p < 0.05 vs. control; α p < 0.05 vs. Lipoic Acid 50 µM; β p < 0.05 vs. VitD3; γ p < 0.05 vs. glutathione; δ p < 0.05 vs. NAC 12 mM.

Article Snippet: The Human Phospho-Src (Y419) Duo set ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to measure phospho-Src in HepG2 cell lysate according to the manufacturer’s instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell reports

Article Title: Small-molecule and CRISPR screening converge to reveal RTK dependencies in pediatric rhabdoid tumors

doi: 10.1016/j.celrep.2019.07.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ponatinib (ABL1;PDGFRA;KDR;FGFR1;SRC;KIT) , SelleckChem , S1490; CAS: 943319-70-8.

Techniques: Recombinant, Cell Viability Assay, Protease Inhibitor, Injection, Sample Prep, Expressing, Activity Assay, Plasmid Preparation, Software, Quantitative Proteomics, Methylation, Membrane