Immunofluorescence staining and confocal microscopy

Cells were seeded onto 8-well glass slides at a density of 1.5 × 10⁴ cells/well and incubated overnight in the presence of 0.1 µg/ml (isoform 1) or 1 µg/ml (EV and isoform 2) doxycycline. 24 h post-induction, the culture medium was replaced and cells were treated with either 100 nM Bafilomycin (Santa Cruz Biotechnology) or vehicle control (DMSO) for 4 h. Subsequently, cells were fixed with 4% formaldehyde (Thermo Scientific) and incubated with blocking buffer (5% normal goat serum/0.3% Triton X-100 in PBS) for 1 h at room temperature. Following staining with primary antibodies diluted in PBS containing 1% BSA and 0.3% Triton X-100 overnight at 4 °C, cells were incubated with Alexa Fluor 546 or 555 anti-mouse or anti-rabbit IgG secondary antibodies (Life Technologies) for 1 h at room temperature and counterstained with DAPI (Invitrogen, Carlsbad, CA). Finally, slides were mounted in Fluoroshield (Sigma-Aldrich) and images were acquired on a confocal microscope (Olympus FLV1200). Representative images of at least three independent experiments are shown. LC3B puncta were quantified using CellProfiler software and the number of puncta was normalized to the number of nuclei.