Skip to main content
. 2018 Feb 28;22(9):2395–2407. doi: 10.1016/j.celrep.2018.02.024

Figure 4.

Figure 4

Induction of Autophagy by Acetylsalicylic Acid In Vivo

(A and B) Representative immunoblots (n = 3 mice per condition, n = 3 experiments) showing LC3I-to-LC3II conversion and depletion of the autophagic substrate SQSTM1/p62 in the heart (A) and in the liver (B) (1, 3, and 6 hr after intraperitoneal (i.p.) injection of 100 mg/kg aspirin). Autophagy induction in vivo is paralleled by a transient decrease in H3K56 acetylation, precedes PRKAA activation (as monitored by upstream kinase-dependent phosphorylation on Thr172 and an increase in PRKAA-mediated ACACA phosphorylation on Ser79), and it is accompanied by a reduction in the mTORC1 substrate PRS6K1. GAPDH levels were monitored to ensure equal protein content. Quantification of (A) and (B) are shown in Figures S3A–S3F and S4A–S4F, respectively.

(C and D) Representative immunoblots (n = 2 experiments, n = 3 mice per group) showing LC3I-to-LC3II conversion in the presence or absence of the protease inhibitor leupeptin (Leup) in the heart (C) and liver (D) 6 hr after aspirin treatment. Quantifications are presented in Figures S3G and S4G, respectively.

(E and F) Long-term aspirin treatment enhances autophagic flux in the heart and stimulates mitophagy.

(E) Transgenic mice expressing the tandem-fluorescent mRFP-GFP-LC3 (Tg-tf-LC3) were treated for 2 weeks with 25 mg/kg aspirin by oral gavage, and autophagic flux was assessed by the injection of chloroquine (10 mg/kg) 4 hr prior to euthanasia. Representative images of fluorescent GFP-LC3 puncta and mRFP-puncta are shown in the left panel. Arrows, autophagosomes; arrowheads, autolysosomes. Values in the right panel represent mean number of autophagosomes (yellow bars) and autolysosomes (red bars) per cell ± SD from one representative experiment (∗∗∗p < 0.001 and ##p < 0.01, one-way ANOVA compared to respective control conditions).

(F) Evaluation of mitochondrial autophagy by transgenic Mito-Keima mice. 2-month-old C57BL/6J mouse hearts were examined in the control versus aspirin-treated group (n = 3 mice/group). Representative images of Mito-Keima green (457 nm), Mito-Keima red (561 nm), the merged image, and a ratiometric image of red-to-green Mito-Keima (561/457 nm) indicating mitophagy are shown (left panel) and quantified (right panel). Values represent means ± SD from one representative experiment (∗∗∗p < 0.001, unpaired t test compared to control group). Scale bar, 50 μm.