TABLE 1

Expression of dominant-negative PERK or treatment with 3MA suppresses the formation of GFP-LC3 containing vacuoles after OSU-03012 exposure

U251 cells were plated in four-chamber glass slides and 24 h later transfected with an empty vector plasmid or a plasmid to express a GFP-tagged form of LC3. In parallel, the cells were transfected with an empty vector plasmid or a plasmid to express a truncated dominant negative form of PERK. Twenty-four hours after transfection, cells were pretreated with vehicle (veh, PBS) or 3MA (5 mM) followed 30 min later by vehicle (VEH, DMSO) or with OSU-03012 (OSU, 1 μM). Six hours after OSU-03012 treatment, cells were visualized at 40× using an Axiovert 200 fluorescent microscope under fluorescent light, on the Zeiss Axiovert 200 microscope using the FITC filter, and under visible light. Data shown is from 40 transfected cells from a representative experiment (n = 3). Mean autophagic vesicles per U251 cell 24 h after treatment (n = 40 LC3.GFP-transfected cells ± S.E.M.).