The MVEC line EOMA was purchased from ATCC and cultured as described [26 (link)]. In brief, MVECs were maintained in DMEM (Thermo, USA) supplemented with 10% FCS (Thermo, USA) and 1% penicillin/streptomycin (Thermo, USA), and incubated at 37°C and 5%CO2. Cells were treated with indicated dose of amitriptyline, 10 nM bafilomycin B1 (Sigma, MO, USA), 5 μM chloroquine (Sigma, MO, USA) or 10 μM spautin-1 (Sigma, MO, USA) for 6 hr.
Spautin 1
Manufactured by Merck Group
Sourced in United States, Germany
Spautin-1 is a small molecule compound that inhibits the activity of the proteins ULK1 and ULK2. These proteins play a key role in the initiation of autophagy, a cellular process by which damaged or unwanted components are broken down and recycled.
Automatically generated - may contain errors
Lab products found in correlation
Bafilomycin a1, by Merck Group (7 mentions)
Chloroquine, by Merck Group (5 mentions)
Rapamycin, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Necrostatin 1, by Merck Group (2 mentions)
Sbi 0206965, by Merck Group (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Cisplatin, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Niclosamide, by Merck Group (2 mentions)
3 methyladenine, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
29 protocols using spautin 1
1
Amitriptyline and Autophagy Modulation
2
In vitro RCC Cell Line Characterization
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Human RCC cell lines, characterized as an adenocarcinoma (ACHN) and clear cell carcinomas (Caki-1 and 769-P), were originally purchased from ATCC [25 –27 (link)]. All cells were cultured in RPMI with 10% FBS, 1% glutamine, and 1% Pen/Strep. For in vitro experiments, cells were seeded on the appropriated plates overnight and treated with HCQ (75 or 100 μM, Acro Chemicals), RAD001 (10 μM, LC Laboratories), bafilomycin A1 (50 nM, Sigma), or spautin-1 (10 μM, Sigma) for 48 hours. Bortezomib (Velcade, 1 μ g/ml) was obtained from the Fox Chase Cancer Center pharmacy and treated cells for 16 hours.
3
EGFR TKIs Modulate Autophagy
4
BKPyV Infection and Autophagy Modulation
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5×103 cell were plated in 96-well culture plates. The following day, cells were treated with multiple drugs that influence autophagy levels, rapamycin, 3-methyladenine, bafilomycin-A, DMSO, and spautin-1 (Sigma) before and after being infected with BKPyV (MOI:5) for 1 h at 37 °C. 72 h post infection, cells were fixed using 2% paraformaldehyde (EMB) and permeabilized using 0.5% Triton X-100 (Shelton Scientific). To detect the number of infected cells expressing viral protein, cells were incubated with the anti-V antigen (V-Ag) monoclonal antibody PAB597, secondary Alexa-Fluor-488-labeled goat anti-mouse antibody (Molecular Probes), and then counterstained with DAPI-mounting media (Vector Labs). The PAB597 hybridoma produced a monoclonal antibody against the SV40 major capsid protein VP1 that cross-reacts with BKPyV VP1. Infected cells were scored using a Nikon epifluorescence microscope (Eclipse E800; Nikon, Inc.). Approximately 20,000 cells, the entire surface of the 96 well, were screened for V-Ag expression.
5
Deciphering Cell Death Mechanisms
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To determine cell death mechanism, we used different inhibitors: N-acetyl-cysteine (NAC, 5mM; Sigma-Aldrich, St Louis, MO, USA) as ROS inhibitor,12 (link) QVD-OPh (QVD; 10 μM; BioVision, Milpitas, CA, USA) as pan-caspase inhibitor,15 (link) Spautin-1 (0.75 μM; Sigma-Aldrich, St Louis, Missouri, USA) as an autophagy inhibitor,16 (link) and Necrostatin-1 (5 μM; Sigma-Aldrich, St Louis, Missouri, USA) as necroptosis inhibitor.17 (link) The inhibitors were added 30 mins before treatment with CH-AuNPs. All stock solutions were wrapped in foil and stored at −20°C.
6
Dendritic Cell Infection Dynamics
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DCs (2 × 106) were infected in a total volume of 300 µl RPMI 1640 medium supplemented with 20 mM Hepes at an MOI of 2. Infection was performed for 1 h at 37°C at 300 rpm in a shaking heating block (Eppendorf). Subsequently, cells were transferred to RPMI 1640 medium containing 1% autologous serum, 10 mM Hepes, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 40 U/ml GM-CSF, and 250 U/ml IL-4, adjusted to a final concentration of 106 cells/ml.
Where indicated, cells were treated with 10 µM MG-132, 2 µM bortezomib, or 10 nM rapamycin (all from Enzo Life Sciences) at 1 hpi. To inhibit autophagy and lysosomal degradation, cells were incubated with 10 µM spautin-1 or 1 µM BA-1 (all from Sigma-Aldrich) 1 h before infection.
Where indicated, cells were treated with 10 µM MG-132, 2 µM bortezomib, or 10 nM rapamycin (all from Enzo Life Sciences) at 1 hpi. To inhibit autophagy and lysosomal degradation, cells were incubated with 10 µM spautin-1 or 1 µM BA-1 (all from Sigma-Aldrich) 1 h before infection.
7
Primary Keratinocyte Isolation and Culture
8
Antibody-Based Autophagy and Apoptosis Analysis
9
Autophagy modulators in cell signaling
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BafA1 was obtained from Wako Chemicals or AdipoGen Life Sciences. Concanamycin A was obtained from Santa Cruz Biotechnology. Pepstatin A, E-64-d and leupeptin were obtained from Peptide Institute (Osaka, Japan). Wortmannin and MG-132 were obtained from Calbiochem. Ammonium chloride, novobiocin sodium salt, 3-MA, spautin-1 and 6-aminonicotinamide were obtained from Sigma. Recombinant mouse IL-1β was obtained from Cell Signaling Technology. The TLR2 ligand FSL-1 was obtained from InvivoGen. Other reagents were obtained from Wako Chemicals.
10
Osteogenic Differentiation of HGMSCs
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HGMSCs were cultivated under standard conditions (37 °C, 95% air: 5% CO2 v/v) in α-Minimum Essential Medium Eagle (α-MEM, Cod. M8042, Sigma-Aldrich, St. Luis, MO, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, cod. ECS0180L; Euroclone S.p.A., Milan, Italy), 2 mM L-glutamine (cod. G7513, Sigma-Aldrich) and 1% w/v of Penicillin/Streptomycin (cod. P0781, Sigma-Aldrich). For osteogenic differentiation, cells were incubated up to 21 days in α-MEM supplemented with 50 μg/mL of L-ascorbic acid 2-phosphate (Cod. 49,752, Sigma-Aldrich), 100 nM of dexamethasone (Cod. D1756, Sigma-Aldrich), and 10 mM of β-glycerophosphate (Cod. G9422, Sigma-Aldrich) (referred to as ‘differentiation medium’) [30 (link)]. Differentiation medium was replaced twice or thrice a week by adding all previous reagents. Resveratrol (RV, Cod. R5010, Sigma-Aldrich) was added to the standard or differentiation medium as indicated. Where reported, 5 μM spautin-1 (Sp1, Cod. SML0440, Sigma-Aldrich) was added to the culture medium.
HGMSCs were seeded on 35 mm Petri dishes at 80.000 cells per dish or on sterile glass coverslips at 10.000 cells per dish for western blot and immunofluorescence analysis, respectively. For histochemical staining with Alizarin Red Staining, the cells were cultured on 24-well plates at 20.000 cells per well and let adhere 24 h before treatments.
HGMSCs were seeded on 35 mm Petri dishes at 80.000 cells per dish or on sterile glass coverslips at 10.000 cells per dish for western blot and immunofluorescence analysis, respectively. For histochemical staining with Alizarin Red Staining, the cells were cultured on 24-well plates at 20.000 cells per well and let adhere 24 h before treatments.
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