Journal: Frontiers in Endocrinology

Article Title: Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line

doi: 10.3389/fendo.2019.00682

Figure Lengend Snippet: Hanging drop method. BON1 cells were seeded into a 96 well hanging drop plate and treatments with Sunitinib were performed after spheroids transfer in a regular 96 well plate. (A) In the upper lane (Day 3) pictures were taken at day 3 after seeding inside the 96 hanging drop plate with EVOS FL Cell imaging System (10 × objective); in the lower lane (Day 7) spheroids pictures were taken at day 7 after transfer in a regular 96 well plate. Spheroids were treated at day 3, after transfer from the first to the second plate, with Sunitinib 2.5, 5, and 7 μM. (B) Perimeter analysis of spheroids was performed at day 3 and 7 and represented in a graph. Gray column: perimeter analysis at Day 3, before treatments. White columns: perimeter analysis at Day 7 under indicated treatments. The analysis was performed using Image J software and measurements were performed from three independent experiments in two replicates. (C) Cell viability was measured as absorbance in three independent experiments with six replicates each, and it is expressed as the mean ± S.E.M. * P < 0.05 vs. vehicle cells.

Article Snippet: Sunitinib was purchased from Selleckchem (TX, USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −80°C as 10 mM stock solution until use.

Techniques: Imaging, Software

Journal: Frontiers in Endocrinology

Article Title: Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line

doi: 10.3389/fendo.2019.00682

Figure Lengend Snippet: 24-well plate with a cell-repellent surface method. BON1 cells were seeded into a 24 well plate with a repellent surface, mixed overnight at 80 rpm. (A) Spheroids were treated with increasing Sunitinib concentrations and pictures were taken at day 4, 7, and 10 after seeding with a Zeiss Axiovert 200/M-based phase-contrast microscope (5 × objective). (B) Perimeter analysis of spheroids was performed at day 4, 7, and 10. Gray column: perimeter analysis at Day 4, before treatments. White columns: perimeter analysis at Day 7 and 10 under indicated treatments. The analysis was performed using Image J software and measurements were performed evaluating three independent experiments in two replicates. ** P < 0.01 vs. vehicle cells at Day 10. § = 5 μM measurement was not detectable for technical reasons, as indicated in the results section. (C) Immunohistochemical expression of Caspase 3 in spheroids treated with different Sunitinib concentrations. Spheroids were fixed at day 10 and pictures were taken with a Zeiss Axiovert 200/M-based phase-contrast microscope. Pictures provide an overview of the entire spheroid stained with eosin and Caspase 3 antibody.

Article Snippet: Sunitinib was purchased from Selleckchem (TX, USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −80°C as 10 mM stock solution until use.

Techniques: Microscopy, Software, Immunohistochemical staining, Expressing, Staining

Journal: Frontiers in Endocrinology

Article Title: Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line

doi: 10.3389/fendo.2019.00682

Figure Lengend Snippet: ULA plate method. BON1 cells were seeded into an ultra low attachment 96 well plate and spheroids were obtained by centrifugation. (A) Spheroids were treated with increasing Sunitinib concentrations and pictures were taken with EVOS FL Cell imaging System (10 × objective) at Day 3 and 7 after seeding. (B) Perimeter analysis of spheroids was performed at Day 3 and 7. Gray column: perimeter analysis at Day 3, before treatments. White columns: perimeter analysis at Day 7 under indicated treatments. The analysis was performed using Image J software and measurements were performed from three independent experiments in two replicates. * P < 0.05 vs. vehicle cells. (C) Cell metabolic activity was measured as absorbance in three independent experiments with six replicates each, and it is expressed as the mean ± S.E.M. ** P < 0.01 vs. vehicle cells.

Article Snippet: Sunitinib was purchased from Selleckchem (TX, USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −80°C as 10 mM stock solution until use.

Techniques: Centrifugation, Imaging, Software, Activity Assay

Journal: PLoS Pathogens

Article Title: Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness

doi: 10.1371/journal.ppat.1007276

Figure Lengend Snippet: (A) HEK 293 cells were transfected with 1 μg of (i) EGFP or (ii) EGFP-ST expression plasmids and grown in the absence or presence of GI254023X or TAPI-2 inhibitors. 24 hours later cells were fixed and EGFP fluorescence analysed by direct visualisation, whereas endogenous Alpha-E-Catenin was identified by indirect immunofluorescence using a specific antibody. The top panel for both (i) and (ii) is the same as . (B) EGFP or EGFP-ST transfected HEK 293 cells were grown in the absence or presence of GI254023X or TAPI-2 inhibitors for 24 hours, then harvested and stained with an Alpha-E-catenin specific antibody and Alexa-Fluor-tagged secondary antibody. Mean fluorescence intensity was analyzed using FlowJo software. Fold difference of cell surface staining was calculated using three replicates per experiment, n = 3 by a two-tailed t-test with unequal variance, *** = p<0.001 and ** = p<0.01.

Article Snippet: ADAM 10 specific inhibitor, GI254023X and ADAM 10/17 dual inhibitor, TAPI-2 where purchased from TOCRIS and Merck Millipore, respectively.

Techniques: Transfection, Expressing, Fluorescence, Immunofluorescence, Staining, Software, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness

doi: 10.1371/journal.ppat.1007276

Figure Lengend Snippet: (A) EGFP or EGFP-ST transfected HEK 293 cells were incubated with the ADAM 10 specific inhibitor, GI254023X (50 μM), then serum starved for 24 hours to induce aggregate formation. Upon reintroduction of serum, cells were fixed and stained with DAPI at 24 hourly intervals. Images were analysed using Image-J to quantify the distance between each cell nucleus. Data analysed using three replicates per experiment, n = 50 cells, by a two-tailed t-test with unequal variance, **** = p≤ 0.0001. (B) HEK 293 cells were transfected with 1 μg EGFP or EGFP-ST in the presence of either scramble or ADAM 10-specific siRNAs. After 24 hours, cell lysates were probed using ADAM 10- and Alpha-E-catenin specific antibodies. GAPDH was used to measure equal loading. 2T2 was used to probe for MCPyV ST expression. (C) Densitometry of immunoblots was performed using ImageJ software. Data analysed using three replicates per experiment, n = 3 and statistical analysis using a two-tailed t-test with unequal variance, *** = p<0.001. (D) HEK 293 cells were transfected with 1 μg EGFP or EGFP-ST in the presence of either scramble or ADAM 10-specific siRNAs, then serum starved for 24 hours to induce aggregate formation. Upon reintroduction of serum, cells were fixed and stained with DAPI at 6 hourly intervals. Images were analysed using Image-J to quantify the distance between each cell nucleus. Data analysed using three replicates per experiment, n = 50 cells, by a two-tailed t-test with unequal variance, **** = p≤ 0.0001.

Article Snippet: ADAM 10 specific inhibitor, GI254023X and ADAM 10/17 dual inhibitor, TAPI-2 where purchased from TOCRIS and Merck Millipore, respectively.

Techniques: Transfection, Incubation, Staining, Two Tailed Test, Expressing, Western Blot, Software

Journal: PLoS Pathogens

Article Title: Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness

doi: 10.1371/journal.ppat.1007276

Figure Lengend Snippet: (A) EGFP or EGFP-ST transfected (i) HEK 293 or (ii) MCC13 cells were incubated with DMSO or the ADAM 10 specific inhibitor, GI254023X (50 μM). (B) EGFP or EGFP-ST transfected (i) HEK 293 or (ii) MCC13 cells were incubated with DMSO or the ADAM 10/17 dual inhibitor, TAPI-2 (50 μM). (C). HEK 293 cells were transfected with 1 μg EGFP or EGFP-ST in the presence of either scramble or ADAM 10-specific siRNAs. After 24 hours, cell motility was analysed using an IncuCyte Zoom-kinetic live cell imaging system. Images were taken every 30 minutes for a 24 hour period. The movement of cells were then tracked using Image J software and the average distance travelled was measured in μm (n = 50 per condition) and significance was tested using a 3-tailed Student’s t-test, *** = p<0.001 and ** = p<0.01.

Article Snippet: ADAM 10 specific inhibitor, GI254023X and ADAM 10/17 dual inhibitor, TAPI-2 where purchased from TOCRIS and Merck Millipore, respectively.

Techniques: Transfection, Incubation, Live Cell Imaging, Software

Journal: PLoS Pathogens

Article Title: Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness

doi: 10.1371/journal.ppat.1007276

Figure Lengend Snippet: (A) MCPyV positive MCC cell lines, PeTa and WAGA, were incubated with DMSO or the ADAM 10 specific inhibitor, GI254023X (50 μM). Cells were then transferred into migration wells and allowed to migrate from serum-free to 10% FBS conditions for 24 hours. Migratory cells were stained and measured at 560 nm to quantify migration. Average cell migration was calculated and significance tested using a two-tailed Student’s t-test (n = 3), *** = p<0.001. (B) (i) PeTa and WAGA cells were transfected with either scramble or ADAM 10-specific siRNAs and cell lysates probed to confirm successful knockdown with an ADAM 10-specific antibody. GAPDH was used as loading control. 2T2 was used to probe for MCPyV ST expression. (ii) Control and ADAM 10-depleted cells were then transferred into migration wells and allowed to migrate from serum-free to 10% FBS conditions for 24 hours. Migratory cells were stained and measured at 560 nm to quantify migration. Average cell migration was calculated and significance tested using a two-tailed Student’s t-test (n = 3), **** = p<0.0001, *** = p<0.001.

Article Snippet: ADAM 10 specific inhibitor, GI254023X and ADAM 10/17 dual inhibitor, TAPI-2 where purchased from TOCRIS and Merck Millipore, respectively.

Techniques: Incubation, Migration, Staining, Two Tailed Test, Transfection, Knockdown, Control, Expressing

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PKI 14–22 amide, myristoylated , Tocris, Bio-Techne , 2546.

Techniques: Virus, Recombinant, Cell Culture, Saline, Sterility, Live Cell Imaging, Transfection, Protein Extraction, Protease Inhibitor, Labeling, SYBR Green Assay, Ointment, Adhesive, Gel Extraction, Isolation, TA Cloning, Sequencing, Plasmid Preparation, shRNA, Software, Control

Journal: Cell

Article Title: FXR regulates intestinal cancer stem cell proliferation

doi: 10.1016/j.cell.2019.01.036

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: glycolithocholic acid (GLCA) , Steraloids , Cat# C1437-000.

Techniques: Generated, Recombinant, Acid Assay, Bioassay, Enzyme-linked Immunosorbent Assay, Cell Viability Assay, Viability Assay, Isolation, Flow Cytometry, Imaging, In Vivo, Reverse Transcription, SYBR Green Assay, Western Blot, Gentle, Sequencing, Software