Fig. 6.

Blocking proteasome activity reverses PTEN stability and activation of autophagy in ATGL KO mice and cardiomyocytes. a, WT and ATGL KO mice were subjected to sham or TAC operation for 4 weeks. Measurement of the proteasome caspase-like, trypsin-like, and chymotrypsin-like activities in the heart (n = 8). b, Immunoblotting of the proteasome catalytic subunits (β1i, β2i, and β5i) in the heart (left), and quantification of the relative protein levels (right, n = 4). c, Immunoblotting of PTEN, p-AKT, AKT, Sirt1 and GAPDH in the heart (left). Quantification of the relative protein levels (right, n = 4). d, WT and ATGL KO mice were subjected to TAC operation and co-treated with VO-OHpic (10 mg/kg daily) or epoxomicin (2.9 mg/kg daily) continuously for 4 weeks. Immunoblotting analysis of PTEN, p-mTOR, mTOR, LC3, and GAPDH in the heart (left). Quantification of the relative protein levels (right, n = 4). e, Neonatal rat cardiomyocytes (NRCMs) were co-infected with adenovirus containing mRFP-GFP-LC3 and Ad-siRNA-control or Ad-siRNA-ATGL, and then treated with Ang II (100 nM) in the presence or absence of VO-OHpic (50 nM) or epoxomicin (100 nM) continuously for 48 h. Immunofluorescence images (left) and the numbers of autophagosomes (yellow) and autolysosomes (red) in each condition (right, 15–20 cells per group) were quantified (n = 5 independent experiments). Scale bar: 20 μm. Data are presented as mean ± SEM, and n represents number of animals