Fig. 5.

BRUCE controls autolysosome formation. a Immunofluorescent staining of endogenous LC3B and LAMP2 in Bruce^+/+ and Bruce^−/− MEFs. Bruce^+/+ and Bruce^−/− MEFs in fully supplemented medium (FM), starved (Starv) for 4 h with or without Bafilomycin A1 (Baf; 100 nM) were fixed and stained with anti-BRUCE and anti-LAMP2 antibodies. Scale bars, 20 µm. b Percentage of LAMP2-positive vesicles containing LC3B-positive aggregates in MEFs under starvation condition with Bafilomycin A1 treatment as in a. Data are presented as dot plots with mean±SD (***p < 0.001, analyzed by t-test); n = 10 cells. c Electron microscopic images of Bruce^+/+ and Bruce^−/− MEFs in FM or starved for 2 h. Overview: scale bars, 10 µm; magnified: scale bars, 2 µm. Green and pink arrows point to autophagosomes and autolysosomes, respectively. d Examination of the endosomal/lysosomal pathway by confocal microscopy using DQ-Ovalbumin in Bruce^+/+ and Bruce^−/− MEFs. Cells were grown in DQ-Ovalbumin containing starvation media for the indicated time. Scale bars, 20 µm