Autophagy Inhibitor, 3-MA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
It interferes with LC3B-I-LC3B-II conversion and inhibits autophagosome formation.	Use 5mM concentration of 3-MA to treat the cells

Upstream tips
It interferes with LC3B-I-LC3B-II conversion and inhibits autophagosome formation.
Protocol tips
Use 5mM concentration of 3-MA to treat the cells

Publication protocol

Ganoderic acid DM and cellular cytotoxicity assay
Ganoderic Acid DM (GA-DM), originally isolated from the Ganoderma lucidum mushroom, was purchased from Planta Analytica, LLC (Danbury, CT) (Cat# G-032). The purity of GA-DM was determined by the vendor as 99.9% using LC/MS analysis. GA-DM was dissolved in DMSO (Sigma) to make a 10 mM stock solution for use in the cytotoxicity assay. For all GA-DM treatments, DMSO final concentration was ≤1%. Melanoma cell lines J3, HT-144, 1359-mel, and DM-331 were seeded at 1×104 cells/well in 100 μl of appropriate culture medium in a flat-bottom 96-well plate. GA-DM was then added to appropriate wells for final concentrations of 0, 10, 20, 30, 40, 50, 60 and 80 μM for 24 h at 37°C. Cells (J3 and HT-144) were also treated with 40 μM of GA-DM for 3, 6, 12, and 24 h, in the presence or absence of an autophagy inhibitor [3-methyladenine (3-MA, 5mM)] (Sigma) or a pan caspase inhibitor (Z-VAD-FMK, 50 μM) (R&D systems #FMK001, Minneapolis, MN) for overnight. Following treatment, cell viability was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI) [28]. Twenty μl of MTS reagent was added to each well, and the plate was incubated for 2 h at 37°C. After incubation, absorbance was read at 490 nm. Cells treated with vehicle alone were used as controls. The percent cell death induced by GA-DM was calculated using the equation: [ (Absorbance control - Absorbance treated)/Absorbance control] ×100. Experiments were repeated at least three times and the data were expressed as percent cytotoxicity ± S.D. of triplicate wells.

Western blot analysis
J3.DR4 and HT-144 cells were cultured for 24 h in the presence of vehicle alone or 40 μM of GA-DM. Following treatment, cells were washed and cell lysate was obtained using a standard lysis buffer (10 mM Trizma base, 150 mm NaCl, 1% Triton-X 100) [16, 28]. Equal protein concentrations from designated cell lysates were separated on a 4-12% Bis/Tris NuPage gel (Invitrogen, Grand Island, NY). Proteins were transferred onto a nitrocellulose membrane (Pierce, Rockford IL), and probed with antibodies for the expression of caspase 3 and 9, cytochrome c, Apaf-1, survivin, Bcl-2, Bax, Beclin-1, LC3, HLA class II (HLA-DR), HLA-DM, invariant chain (Ii), and LAMP-2 proteins [31, 32]. The secondary antibodies used were horseradish peroxidase conjugated anti-mouse (Pierce), anti-rabbit or anti-goat IgG (Santa Cruz Biotechnology). Monoclonal antibody for β-actin was used as a protein loading control.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - J3 using Autophagy Inhibitor, 3-MA from Sigma-Aldrich.

Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Autophagy Inhibitor, 3-MA below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - J3 using Autophagy Inhibitor, 3-MA from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.