1. Autophagy protein abundance, but not autophagy flux, is altered in Cnot3-depleted heart.
Autophagy protein abundance, but not autophagy flux, is altered in Cnot3-depleted heart. (A) Transmission electron microscope (TEM) analysis for WT (representative of n = 3) and Cnot3 mKO (representative of n = 3) mouse hearts. Higher magnification of the colored rectangles (top) is shown in the bottom panel. Scale bars, 5 μm (top) and 1 μm (bottom). (B) Western blot for autophagy factors in the hearts of WT and Cnot3 mKO mice under fed and fasting conditions. AU, arbitrary units. Representative blots (left and fig. S3A) of n = 3 independent experiments are quantified for fed condition with WT (n = 5) and Cnot3 mKO (n = 5) mice (top right) and for fasting condition with WT (n = 3) and Cnot3 mKO (n = 3) mice (bottom right). (C) Western blot for LC3 (microtubule-associated protein 1 light chain 3) and p62 in the hearts of WT and Cnot3 mKO mice treated with or without bafilomycin A1 (Baf-A1). Representative blots (left and fig. S3C) of n = 3 independent experiments were quantified for WT mice treated with vehicle (n = 7), Cnot3 mKO mice treated with vehicle (n = 7), WT mice treated with Baf-A1 (n = 6), and Cnot3 mKO mice treated with Baf-A1 (n = 6). (D) Immunocytochemistry of LC3 in mouse cardiomyocytes transfected with Cnot3 (si-Cnot3) or control (si-Control) small interfering RNAs (siRNAs) and treated with or without E64d and pepstatin A (Pep) [n = 2 independent experiments with two different siRNAs for Cnot3 (fig. S3D)]. Scale bars, 20 μm. (E) Western blot for LC3 and p62 in WT and Cnot3 KO mouse embryonic fibroblasts (MEFs) treated with or without E64d plus pepstatin A (Pep) (n = 2 independent experiments). All values are means ± SEM. *P < 0.05, unpaired two-tailed Student’s t tests.
Tomokazu Yamaguchi et al., Sci. Signal. 2018;11:eaan3638
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