Figure 3.

Chloroquine enhances secretion of TAX1BP1 in an ATG16L1-dependent manner. (A) Whole cell pellet collected alongside the secreted fraction were lysed and probed for TAX1BP1, GABARAP, and LC3B. Significance was determined by paired t-test (*: p < 0.05; **: p < 0.01; n = 3). ±: plus or minus 10 µM CQ. (B) Secreted particles (SE) were collected from cells treated with or without 10 µM CQ and probed for TAX1BP1, TSG101, GABARAP and LC3B. Equal amount of protein, as determined by BCA assay, was loaded in each lane. (C) CRISPR-mediated knockout (KO) of ATG16L1 with two different single guide RNAs was used to generate isogenic ATG16L1 KO lines in MDA-MB-231 (n = 3). Secreted particles were collected as described and probed for LC3B, TSG101 and TAX1BP1. (D) Isogenic ATG4B KO cells were generated using CRISPR-mediated knockout (KO). Secreted particles were collected from ATG4B KO cells in the presence or absence of 10 µM CQ treatment and probed for LC3B and GABARAPL2 (n = 3). (E) The effect of ATG16L1 knockout and rescue on the lipidation of LC3B and GABARAP in whole cell (WC) and secretion (SE) of LC3B and TAX1BP1 (n = 3). Samples from the same experiment were run on parallel blots. WT: wild type; -: ATG16L1 KO, α: ATG16L1-α rescue of ATG16L1 KO; β: ATG16L1-β rescue of ATG16L1 KO. (F) Densitometry quantitation of SE fraction in (E). Significance was determined by one-way ANOVA with Sidak’s multiple comparison test (***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: not significant; n = 3).