Evaluating ANAVEX2-73 and PRE-084 in HeLa and HEK293A Cells

HeLa and HEK293A cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA, 41965062) supplemented with active FBS (Life Technologies GmbH, Carlsbad, CA, USA, 10270106), 1× ABAM (Invitrogen, 15240-062) and 1 mM sodium pyruvate (Invitrogen, 1136-088). After medium change, they were treated for 2 h with 10, 1, and 0.1 µM ANAVEX2-73 and PRE-084 (Tocris, Bristol, UK, 0589), respectively; ANAVEX2-73 was provided by ANAVEX Life Sciences Corp, New York, NY, USA. Afterwards Bafilomycin A₁ (BafiA₁) (Toronto Research Chemicals, North York, ON, Canada, B110000) was added for a further 2 h and the cells were eventually harvested. Western blot analyses were performed as described previously [40 (link),41 (link)]. Briefly, cells were subjected to SDS–PAGE using precast NuPAGE 4%–12% Bis-Tris gels (Invitrogen, NPO322). Proteins were detected by chemiluminescence using the Amersham Imager 600 (GE).
Confocal fluorescence microscopical analyses of HEK293A cells stably expressing GFP-LC3B (kind gift of Dr. Sharon Tooze) were performed with the laser scanning microscope LSM 710 (Zeiss, Oberkochen, Germany).