Anti lc3 pab

Cells were treated as described [6 (link)] and probed with anti-cleaved PARP (Asp214) PAb (1:500, Cell Signaling Technology, Inc. Danvers, MA, USA); anti-LC3 PAb (1:400, MBL International, Woburn, MA, USA); anti-β-actin MAb (1:10000, MP Biomedicals, Aurora, OH, USA).

Cells were seeded in a 16‐well glass slide (Nunc™ #178599 Lab‐Tek® Chamber Slide™, Austin, USA) at 37°C with 5% CO₂ for 24 h. Autophagosomes were stained with anti‐LC3 pAB (MBL International® #PM036, Massachusetts, USA) and images were taken with a Zeiss LSM confocal microscope (63×). A detailed protocol is presented in the Supporting Information.

To perform fibroblast immunocytochemistry, 5000 cells were counted using the Scepter 2.0 Handheld Automated Cell Counter pipette (Merck) and seeded in a 16-well glass slide (Nunc™ #178599 Lab-Tek^® Chamber Slide™, Austin, TX, USA) at 37 °C with 5% CO₂ for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in a blocking solution (1% bovine serum albumin). Degradation of autophagolysosomes was blocked by adding 100 nM Bafilomycin A1 (Sigma-Aldrich^® #B1793 SIGMA, St. Louis, MO, USA) for 6 h. Autophagosomes were stained with anti-LC3 pAB (MBL International^® #PM036, Woburn, MA, USA) and secondary marked through the donkey anti-rabbit Alexa Fluor^® 488 IgG antibody (Life Technologies Europe, Bleiswijk, Netherlands). Counterstain with DAPI was performed for nuclei staining (DAPI Fluoromount-G^® #0100-20, Southern Biotech, AL, USA). Images were taken with a Zeiss LSM 880 laser scanning confocal system (63×).