Fig. 3.

FUCA1 loss impairs autophagy flux. (A) Immortalized Fuca1^flox/flox MEFs were infected with a retrovirus expressing the Cre recombinase (KO) or a retrovirus control empty vector (WT) and treated with 100 nM BAF or 10 µM CQ for 4 h. Cell lysates were analyzed by immunoblotting using antibodies directed against LC3B and actin, which was used as a loading control. The immunoblot shown is representative of the results observed in three to four independent experiments, and quantified (Right) ± SEM (two-way ANOVA, Sidak’s multiple comparison test: n.s.). LE, long exposure; SE, short exposure. (B) Fuca1^+/+ and Fuca1^−/− MEFs were infected with a retrovirus expressing YFP-Parkin (LZRS-YFP-PARKIN). Cells were then treated with 12.5 μM CCCP for 48 h. Cell lysates were analyzed using the MitoProfile Membrane integrity WB Antibody Mix. Actin was used as a loading control. The immunoblot shown is representative of the results observed in three independent experiments. The percentage of remaining CVa and Core 1 in Fuca1 WT and KO MEFs upon CCCP treatment is quantified ± SEM (two-tailed unpaired t test, CVa *P = 0.021, Core 1 *P = 0.0445). (C) Immortalized Fuca1^+/+ and Fuca1^−/− MEFs were incubated in EBSS for the indicated time period. Cell lysates were analyzed by Western blot using LC3B and actin antibodies. The LC3II : actin ratio is plotted on the right. (D) Primary Fuca1^+/+ and Fuca1^−/− MEFs were incubated in EBSS for 4 h. Electron microscopy images show autophagic degradative vacuoles (red arrows). (Scale bars, 500 nm.)