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 (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.

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