Figure 5.

Autophagosomes not exosomes are the major and potent inducers of IL-1β in DRibbles. (a) A total of 100 μg/ml normal vesicles with no drug treated and bortezomib/NH₄Cl-treated DRibbles from UbiLT3 cells incubated with primed THP-1 overnight. The supernatant IL-1β was tested by ELISA. (b) LC3 status of HEK293 DRibbles and intact cells was measured by western blot and (c) DRibbles labeled with LC3-PE or isotype control was detected by flow cytometry. (d) Intact cells (100 μg/ml) and HEK293 DRibbles (100 μg/ml) incubated with primed THP-1 overnight. The supernatant IL-1β was tested by ELISA. (e) LC3, HSP90 and SGT1 in indicated DRibbles were measured by western blot. (f) Primed THP-1 was stimulated with 5 mM ATP/LPS, 5 μM Nigericin/LPS and 100 μg/ml DRibbles made from indicated cells overnight, the supernatant IL-1β was tested by ELISA. (g) Primed THP-1 was stimulated with UbiLT3 pellet fractions (p300, p1k, p3k, p10k, p30k) at 100 μg/ml overnight, the supernatant IL-1β was tested by ELISA. LC3 status of different cell fractions was measured by western blot. Results are shown as the means+S.D. and representative of two or more independent experiments