Extended Data Fig. 4: Atz-AUTAB treatment damages endosome membrane and triggers LC3C-mediated autophagy.
From: Chemically engineered antibodies for autophagy-based receptor degradation
a,b, HeLa cells stably expressing HA-PD-L1 were treated with 100 nM Atz or Atz-AUTAB for 1 h. Cells were then fixed, co-stained using antibodies against galectin-3, Atz, and Rab5 (a) or Rab7 (b), and imaged by confocal microscopy. Enlarged views of the white boxed regions are shown below. The arrowheads indicate the triple colocalization of galectin-3, Atz-AUTAB, and Rab5 or Rab7. The relative quantification is shown on the right (n = 15 cells per group, means ± s.d.). c,d, HeLa cells concurrently expressing HA-PD-L1 and mCherry-LC3C were treated with 100 nM Atz or Atz-AUTAB for 20 min. After fixation, cells were co-stained with antibodies against Atz and Rab5 (c) or Rab7 (d), followed by confocal microscopy imaging. The white boxed regions are magnified below. The arrowheads indicate the colocalization of mCherry-LC3C and Atz-AUTAB on either early (Rab5-positive) or late (Rab7-positive) endosomes. The quantification is shown on the right (n = 15 cells per group, means ± s.d.). Scale bars, 10 μm. Statistical significance was calculated via unpaired two-tailed Student’s t-test (a-d).
