Figure 6.

Atg8 orthologs are enriched in specific subsets of sEV. (A) Centrifugation density gradient (6% to 30% iodixanol) fractionation of ultrafiltration-concentrated conditioned media from 10 µM CQ treated MDA-MB-231 cells (n = 2). (B) Using anti-CD63 or anti-CD9 antibody-bound magnetic beads, immunoaffinity capture was conducted on pre-cleared and concentrated conditioned media of CQ-treated MDA-MB-231 cells (n = 3). (C) Densitometry quantitation of LC3B and GABARAP levels in CD63-IP were normalized to TSG101 levels in the respective lane. Significance was determined by paired t-test (*: p < 0.05; n = 3). Error bars represent SD. (D) Conditioned media from CQ-treated MDA-MB-231 ATG16L1 KO cells expressing either ATG16L1-α or ATG16L1-β was pre-cleared, concentrated, and incubated with CD63-Dynabeads overnight at 4°C. Beads were washed twice the next day and eluded by boiling in 1X LDS with reducing agent (n = 3). (E) Centrifugation density gradient (6% to 30% iodixanol) fractionation of ultrafiltration-concentrated conditioned media from 10 µM CQ treated MDA-MB-231 ATG16L1 KO cells rescued with either ATG16L1-α or ATG16L1-β (n = 2). (F) Secreted particles (SE) were collected from ATG16L1 KO cells rescued with truncated ATG16L1 (aa 1–249) treated with control or 10 µM CQ. Significance was determined by paired t-test (*: p < 0.05; n = 3). Error bars represent SD. (G) Using anti-CD63 antibody-bound magnetic beads, immunoaffinity capture was conducted on pre-cleared and concentrated conditioned media of CQ treated ATG16L1 KO cells expressing ATG16L1(1–249) (n = 3). (H) Centrifugation density gradient (6% to 30% iodixanol) fractionation of ultrafiltration-concentrated conditioned media from 10 µM CQ treated MDA-MB-231 ATG16L1 KO cells rescued with ATG16L1 (aa1-249) (n = 2). (I) Centrifugation density gradient (6% to 30% iodixanol) fractionation of ultrafiltration-concentrated conditioned media from 10 µM CQ treated MDA-MB-231 ULK1 KO cells (n = 2).