Journal: Scientific Reports

Article Title: Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

doi: 10.1038/s41598-017-17320-2

Figure Lengend Snippet: EGF and PDGF stimulation induce a transient drop in fluorescence of Eps15-GFP or Hrs-YFP on enlarged endosomes. M1 cells stably expressing Ii and Eps15-GFP or Hrs-YFP were stimulated with EGF/PDGF. Data represents the mean ± s.d of 10 independent experiments. ( a ) The graph shows the fluorescence intensity of endosomal Eps15-GFP after ligand stimulation. P value 0.0003 (***) after EGF and < 0.0001 (****) after PDGF stimulation. ( b ) The graph shows the fluorescence intensity of endosomal Hrs-YFP after ligand stimulation. The P value < 0.0001 (****) both after EGF- and PDGF stimulation. ( c ) Control experiments of M1 cells stably expressing CtEEA1-GFP or Rab5-mCherry after EGF stimulation.

Article Snippet: To calculate the endosomal fluorescence intensity, 10 individual endosomes from 10 independent experiments were measured at the specific time points, before and after stimulation with 100 ng/ml human rec EGF (Bachem AG, Bubendorf, CH) or 60 ng/ml human rec PDGF BB (Bachem AG, Bubendorf, CH).

Techniques: Fluorescence, Stable Transfection, Expressing, Control

Journal: Scientific Reports

Article Title: Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

doi: 10.1038/s41598-017-17320-2

Figure Lengend Snippet: EGF and PDGF stimulation induce a transient change in the binding kinetics of Eps15-GFP and Hrs-YFP. M1 cells were serum starved for 4 hours before the FRAP experiments. Single enlarged endosomes positive for Eps15-GFP or Hrs-YFP were bleached at specific time points (0, 4, 8 and 20 minutes) after EGF/PDGF stimulation. All data represents in this figure is the mean value of 10 independent experiments ± s.d. ( a ) The graph shows the immobile fraction (IF) for Eps15-GFP and Hrs-YFP at specific time points after EGF stimulation. P value < 0.0003 (***) for Eps15-GFP and P < 0.0001 (****) for Hrs-YFP. ( b) Graph showing the T 1/2 recovery for Eps15-GFP and Hrs-YFP at similar time points as in (A). P value < 0.0006 (***) for Eps15-GFP and P value 0.0009 (***) for Hrs-YFP. ( c ) The graph shows the IF for Eps15-GFP and Hrs-YFP at specific time points after PDGF stimulation. P < 0.0001 (****) for Eps15 and P < 0.0001 (****) for Hrs-YFP. ( d ) Graph showing the T 1/2 recovery for Eps15-GFP and Hrs-YFP at specific time points after PDGF stimulation. P value < 0.0001 (***) for Eps15-GFP and P value 0.0001 (***) for Hrs-YFP.

Article Snippet: To calculate the endosomal fluorescence intensity, 10 individual endosomes from 10 independent experiments were measured at the specific time points, before and after stimulation with 100 ng/ml human rec EGF (Bachem AG, Bubendorf, CH) or 60 ng/ml human rec PDGF BB (Bachem AG, Bubendorf, CH).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

doi: 10.1038/s41598-017-17320-2

Figure Lengend Snippet: Change in the binding kinetics is induced by phosphorylation. ( a ) This figure shows the total Endosomal Fluorescence Intensity (tEFI) for Eps15-GFP and Hrs-YFP after EGF stimulation divided into their significant fractions, tIF (dark grey) and tMF (light grey). ( b ) This figure shows the total Endosomal Fluorescence Intensity (tEFI) for Eps15-GFP and Hrs-YFP after PDGF stimulation divided into their significant fractions, tIF (dark grey) and tMF (light grey). ( c ) Analysis of the Eps15-GFP and Hrs-YFP phosphorylation level after EGF stimulation. Eps15-GFP and Hrs-YFP were IP with anti-GFP and the phosphorylation level was detected with an anti-phosphotyrosine antibody. The total protein levels of Eps15-GFP and Hrs-YFP after IP were detected with an anti-GFP antibody. The western blot was repeated 3 times. ( d ) Analysis of the Eps15-GFP and Hrs-YFP phosphorylation level after PDGF stimulation. Eps15-GFP and Hrs-YFP were IP with anti-GFP and the phosphorylation level was detected with an anti-phosphotyrosine antibody. The total protein levels of Eps15-GFP and Hrs-YFP after IP were detected with an anti-GFP antibody. The western blot was repeated 3 times.

Article Snippet: To calculate the endosomal fluorescence intensity, 10 individual endosomes from 10 independent experiments were measured at the specific time points, before and after stimulation with 100 ng/ml human rec EGF (Bachem AG, Bubendorf, CH) or 60 ng/ml human rec PDGF BB (Bachem AG, Bubendorf, CH).

Techniques: Binding Assay, Phospho-proteomics, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

doi: 10.1038/s41598-017-17320-2

Figure Lengend Snippet: Endosomally located phosphorylation deficient Eps15-GFP and Hrs-YFP are unaffected by ligand stimulation. ( A ) montage showing EGF-Alexa-647 internalization and localization to enlarged Eps15Y850F-GFP endosomes. ( B ) A montage showing EGF-Alexa-647 internalization and localization to enlarged HrsY329/334F-YFP endosomes. ( c ) This graph shows the endosomal fluorescence intensity of the Eps15 tyrosine mutant (Eps15Y850F-GFP) after stimulation with unlabelled EGF/PDGF. Data represents the mean value of 10 independent experiments ± s.d. ( d ) This graph shows the endosomal fluorescence intensity of the of the Hrs-YFP tyrosine mutant (HrsY329, 334F-YFP) after stimulation with unlabelled EGF/PDGF. Data represents the mean value of 10 independent experiments ± s.d.

Article Snippet: To calculate the endosomal fluorescence intensity, 10 individual endosomes from 10 independent experiments were measured at the specific time points, before and after stimulation with 100 ng/ml human rec EGF (Bachem AG, Bubendorf, CH) or 60 ng/ml human rec PDGF BB (Bachem AG, Bubendorf, CH).

Techniques: Phospho-proteomics, Fluorescence, Mutagenesis

Journal: Scientific Reports

Article Title: Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

doi: 10.1038/s41598-017-17320-2

Figure Lengend Snippet: The mutants Eps15Y850F-GFP and HrsY329/334F-YFP reduce the EGF-R and PDGF-R degradation. The figure shows an EGF-R degradation assay on M1 cells stably expressing Ii-pMep4, co-transfected with either ( a ) Eps15-GFP, ( b ) Hrs-YFP, ( c ) Eps15Y850F-GFP or ( d ) HrsY329/334F-YFP. The cells were treated with cycloheximide (CHX) and stimulated with unlabelled EGF or PDGF BB. The amount of EGF-R was analysed with an antibody against EGF-R, and the total amount of proteins present on the membrane was detected with antibodies against Eps15/Hrs/GFP. Quantification of EGF-R ( e ) and PGDF-R ( f ) degradation assay are based on three individual experiments.

Article Snippet: To calculate the endosomal fluorescence intensity, 10 individual endosomes from 10 independent experiments were measured at the specific time points, before and after stimulation with 100 ng/ml human rec EGF (Bachem AG, Bubendorf, CH) or 60 ng/ml human rec PDGF BB (Bachem AG, Bubendorf, CH).

Techniques: Degradation Assay, Stable Transfection, Expressing, Transfection, Membrane

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que suppressed the proliferation potential of HCC827 cells. For MTT assays, cells were incubated with Que of 25 μM (Que L), 50 μM (Que M), and 100 μM (Que H) for 96 hours. Every 24 hours, cells were subjected to the testing. For colony formation assays, cells were cultured in medium containing 100 μM Que for 2 weeks. ( A ) Detection results of MTT assays. ( B ) Detection results of colony formation assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Incubation, Cell Culture, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que inhibited the invasion and migration potentials, and the signaling transduction of Src/Fn14/NF-κB pathway in HCC827 cells. For scratch assays, cells were incubated with Que of 100 μM (Que H) for 48 hours. For Transwell assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. For western blotting assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. ( A ) Detection results of scratch assays. ( B ) Detection results of Transwell assays. ( C ) Detection results of western blotting assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Migration, Transduction, Incubation, Western Blot, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Overexpression of Src blocked the anti-NSCLC function of Que in HCC827 cells. Cells transfected with negative control vector (NC) or Src expression vector (Src) were treated with Que of 100 μM (Que H) and subjected to MTT assay for 96 hours (cells were collected every 24 hours), and colony formation was assay at 2 weeks, Transwell assay at 24 hours, and scratch assay at 48 hours. ( A ) Detection results of Src level. ( B ) Detection results of MTT assays. ( C ) Detection results of colony formation assay. ( D ) Detection results of Transwell assay. ( E ) Detection results of scratch assay. # P <0.05 versus Que H group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, Expressing, MTT Assay, Transwell Assay, Wound Healing Assay, Colony Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Que inhibited the growth and metastasis potential of NSCLC in vivo in by inhibiting Src signaling. Mice were injected with different HCC827 cells and administrated with Que of 100 mg/kg body weight for 3 weeks. ( A ) Detection results of tumor volume. ( B ) Detection results of hematoxylin and eosin detection of tumor tissue. ( C ) Detection results of western blotting detection of E-cadherin and N-cadherin. * P <0.05 versus Que H+Src group. Scale bar, 100 μm.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: In Vivo, Injection, Western Blot

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibitory activities of Taiwanese local pomegranate fruit. The ethylacetate, butanol, and water layers extracted from pomegranate peel extract (PEP) were tested for the inhibitory efficacy to T24 ( A ) or J82 ( B ) cells using MTT assay as described in Materials and Methods. PEPE2 and PEPE3 ( C ) were also investigated for the inhibitory effectiveness to T24 or J82 cells. 0.1% ( v / v ) Dimethyl sulfoxide (DMSO)-treated urinary bladder urothelial carcinoma (UBUC) cells were regarded as the solvent control. Each MTT result was the typical data of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: MTT Assay, Solvent, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The propidium iodide (PI) and PI/annexin V analyses of UBUC cells treated with PEPE2. The results of PI analyses were represented in ( A ) the T24 cells and ( B ) the J82 cells. The data of the PI/annexin V measurement were shown in ( C ) T24 cells and ( D ) J82 cells. Each flow cytometry figure was the typical result of three independent experiments. The diagram under the PI/annexin V panel was the results of the three independent experiments. The cell cycles and apoptosis detection of the T24 or J82 cells treated with PEPE2 were measured with PI and PI/annexin V analyses, respectively, using flow cytometry as described in the . The 0.1% ( v / v ) DMSO-treated UBUC cells were implemented as the vehicle control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Flow Cytometry, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The molecular mechanisms of apoptotic pathway evoked in PEPE2-incubated UBUC cells. T24 and J82 cells were treated with 50 and 20 μg/mL PEPE2 respectively. Then the protein levels of ( A ) pro-/cleaved caspase-3; ( B ) pro-/cleaved caspase-8, DR4 and DR5; ( C ) pro-/cleaved caspase-9, Bax and Bcl-2 and ( D ) Bip, VCP and pro- caspase-12 in PEPE2-treated T24 cells were measured using western immunoblotting as described in the supporting information. The 0.1% ( v / v ) DMSO-treated UBUC cells were used as the solvent control; ( E ) The proposed molecular apoptotic pathway provoked in the PEPE2-treated UBUC cells. The immunoblot in each figure was the representative result of at least three independent experiments. The diagram (ratio (mean ± standard deviation (S.D.)) under each immunoblot indicated the ratio of the normalized protein intensity (observed protein/actin) of PEPE2-treated cells at the indicated time interval, divided by that at the 0-h time point. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Incubation, Western Blot, Solvent, Control, Standard Deviation

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibition of the xenografted UBUC growth in nude mice by the treatment of the EtOAc layer. T24 cells were injected s.c. into nude mice and were o.g. administrated with water (control), 2, 5, 10, or 100 mg/kg EtOAc layer as described in Materials and Methods. ( A ) The body weights of the extract-treated mice. *** p < 0.001, ** p < 0.005; ( B ) The effects of the EtOAc layer on tumor growth in the xenografted nude mice. The volumes of tumors from the extract-fed mice were compared to those of the water-fed mice. *** p < 0.001; ( C ) The tumor weight. The tumor weight was measured at the 10th week. *** p < 0.001; ( D ) The typical H/E images (400×) of tumor specimens dissected from xenografted nude mice; ( E ) The representative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) images (400×) of tumor lesions from xenografted nude mice. H&E and TUNEL assays were performed as described in Material and Methods.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Inhibition, Injection, Control, End Labeling, TUNEL Assay