Monitoring Autophagy-Immunofluorescence Staining

To analyze autophagy flux, we followed the Guidelines for the use and interpretation of assays for monitoring autophagy.¹⁶ LC3B Antibody Kit for Autophagy (Invitrogen, MA, USA) was adopted for analysis and 25 µM chloroquine diphosphate (CQ) was used as positive control for artificially generating autophagosomes. A 2 × 10⁵ U-87MG cells per well was cultured in Millicell EZ SLIDE 8-well glass slides and maintained with fresh medium containing different doses of WCE or CQ for 24 hours. Next, cells were washed with 1× PBS and fixed with 4% paraformaldehyde. The cells were then incubated in blocking solution and hybridized with antibodies against LC3B (LC3-II) (Invitrogen, MA, USA) after permeabilization in 0.3% Triton X-100 for 6 minutes. Slides were mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc., MA, USA) after the incubation with Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) antibodies (Invitrogen, MA, USA). Finally, the cells were observed under a ZEISS AXioskop2 fluorescence microscope (Carl Zeiss Microscopy, LLC, NY, USA).