Figure 4
From: Phospholipase D1 deficiency in mice causes nonalcoholic fatty liver disease via an autophagy defect
Autophagy is impaired in the livers of Pld1−/− mice.
(a) Primary hepatocytes were isolated from Pld1+/+ and Pld1−/− littermates, cultured in a serum-fed state, and the cell lysates were analyzed by western blotting. (b) Primary hepatocytes isolated as described in (a) were treated with or without E64D (10 μg/ml) and PepA (10 μg/ml) under either serum-fed or serum-starved conditions for 24 h. The cell lysates were analyzed by western blot. (c) Livers were collected from Pld1+/+ and Pld1−/− littermates were fed HFD for 4 weeks (n = 9–11 per group) under either fed or starved conditions for 24 h. Liver lysates were analyzed by western blot. (d) Livers were collected as described in (c). APs were quantitatively analyzed in transmission electron microscopic images of the liver. (e) Pld1+/+ and Pld1−/− MEF were infected with RFP-GFP-LC3B (60 particles per cell) for 24 h and then assigned to either serum-fed or serum-starved conditions. Representative confocal microscopy images are shown. GFP-negative/RFP-positive and GFP-positive/RFP-positive puncta were counted using ZEN software; more than 80–90 cells were included for each of 3 independent experiments. Scale bar, 20 μm. The data are presented as means ± SE or representative blots from 3 to 5 independent experiments. *P < 0.05 versus Pld1+/+ mice. Abbreviations: GFP, green fluorescence protein; HFD; MEF, mouse embryonic fibroblasts; PepA, pepstatin A; RFP, red fluorescence protein; SE, standard error.
