Cells, fixed with 4% paraformaldehyde and permeabilized by 0.5% (v/v) Triton X-100, were incubated with the following antibodies: rabbit anti-LC3 polyclonal antibody (Abgent), mouse anti-DRAM2 monoclonal antibody (Millipore), mouse anti-mitochondria monoclonal antibody (Millipore) as primary antibodies and AlexaFluor 488 anti-rabbit IgG (Thermo Fisher Scientific) or AlexaFluor 594 anti-mouse IgG (Thermo Fisher Scientific) as secondary antibodies. Finally, samples were counterstained with Hoechst 33258 (Sigma) and then mounted in glycerol/PBS (ratio 1:1, pH 7.4).
Samples were observed and acquired through a scanning confocal laser microscope (Leica Microsystems). For morphometric analyses at least 100 cells were evaluated for each experimental point by analyzing merge images acquired by software package IAS 2000 (Delta Sistemi, Rome, Italy).
Samples were observed and acquired through a scanning confocal laser microscope (Leica Microsystems). For morphometric analyses at least 100 cells were evaluated for each experimental point by analyzing merge images acquired by software package IAS 2000 (Delta Sistemi, Rome, Italy).