Extended Data Fig. 3: Atz-AUTAB accelerates PD-L1 endocytosis.
From: Chemically engineered antibodies for autophagy-based receptor degradation
a, Schematic representation of Atz-AUTAB uptake assay (see Methods section for details). b, Confirmation by western blotting of PD-L1 knockdown in stable expressing HA-PD-L1 HeLa cells. c, HA-PD-L1 expressing HeLa cells with scramble or PD-L1 siRNA were incubated with 1 nM Atz or Atz-AUTAB for 1 h. After fixation, the surface-remaining and internalized Atz/Atz-AUTAB were sequentially stained before or after cell permeabilization. The fluorescence intensities of internalized Atz-AUTAB were quantified, and the data are expressed as each normalized value relative to the scramble group (scramble group, n = 16 cells; siPD-L1 group, n = 14, means ± s.d.). d, HA-PD-L1 expressing HeLa cells were incubated with 1 nM Atz or Atz-AUTAB for 1 h. After fixation, the surface-remaining and internalized Atz/Atz-AUTAB were sequentially stained before or after cell permeabilization. The quantification of the internalized Atz or Atz-AUTAB is shown on the right (Atz group, n = 28 cells; Atz-AUTAB group, n = 22; means ± s.d.). e, HeLa cells stably expressing HA-PD-L1 were subjected to antibody feeding-based internalization assay. Representative examples of internalized PD-L1 and surface-remaining PD-L1 are shown. The quantification of the internalized PD-L1 is shown below as indicated (untreated group, n = 15 cells for 1 h internalization, n = 16 for 2 h; L25K PEI group, n = 14 for 1 h, n = 17 for 2 h; Atz group, n = 27 for 1 h, n = 26 for 2 h; Atz-AUTAB group, n = 18 for 1 h, n = 16 for 2 h; means ± s.d.). DNA in c and d was counterstained with DAPI. Scale bars, 10 μm. Statistical significance was calculated via unpaired two-tailed Student’s t-test (c-e).
