3 methyladenine 3ma

The relative contribution of apoptosis and autophagy to the reduced viability of the cancer cells exposed to FVE was examined using co-treatment with specific inhibitors. The pan-caspase inhibitor, Z-VAD (OMe)-FMK (VAD, 60 μM) and the autophagy inhibitor, 3-methyladenine (3-MA, 8 mM; Cayman Chemicals, Ann Arbor, MI) were used in co-treatments with 1 % FVE -for 72 h.

The BV2 murine microglial cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA), supplemented with 5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA), penicillin (20 U/mL), and streptomycin (20 mg/mL) in a 5% CO₂ incubator. The BV2 cells were stimulated with lipopolysaccharide (LPS, obtained from Escherichia coli O111: B4) at the concentration as indicated in each experiment. For the inhibitor treatment, the BV2 cells were pretreated with Bafilomycin A1 (BA; 50 nM, Sigma, St. Louis, MO, USA), Chloroquine (CQ; 5 μM, Sigma), or 3-methyladenine (3MA; 5 mM. Caymanchem, Ann Arbor, MI, USA) for 2 h and then treated with LPS (1 μg/mL) for 24 h.

For cell treatments, TubAstatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO). Cyclopamine (cyclo; Merck KGaA, Darmstadt, Germany) was resuspended in ethanol. 3-methyladenine (3-MA; Cayman Chemical Co, Ann Arbor MI, USA) was dissolved in pure water. In all of the experiments. the vehicle’s final concentration never exceeded 0.1% (v/v). U87-MG cells were seeded at the density of 3300 cells/well in 96-well plates and treated for 48 h in a single or combination setting and in the presence or absence of the following selected subtoxic doses of TubA (8 μM), cyclo (10 μM), BafA1 (30 μM), and 3-MA (2 mM). In zebrafish, TubA, cyclo, rapamycin (Rap; MedChemExpress, Princeton NJ, USA), and BafA1 were dissolved in DMSO. Treatments were performed in a 24-well plate, with a maximum of 15 embryos/well from the stage at 1 day postfertilization (dpf) to 2 dpf. Tg(CMV:eGFP-map1Lc3b) embryos were treated with 25 μM of TubA and 5 μM of cyclo, alone or in combination setting, in 1 mL of final volume of E3 with PTU 1X. The dose–response assay for TubA and cyclo and the modulation of autophagy with 1 μM of rapamicyn (Rap) or 20 nM of BafA1 are shown in the Supplementary Figure S5.