Figure 2 (See previous page).

Intermittent fasting is associated with impaired autophagic flux in Lamp2 heterozygous null mice. (A) Representative gray-scale images (at 630X magnification) of female Lamp2 heterozygous null and littermate wild-type female mice bearing the GFP-LC3 transgene, after 48 h of fasting or in a fed (nonfasted) state. (B) Quantification of cardiomyocyte GFP-LC3 in Lamp2 heterozygous null mice or littermate control females after fasting and/or refeeding for the indicated duration; n = 3 or 4/group. P values depicted are by post-hoc test after one-way ANOVA. (C) Immunoblots depicting LC3 processing and SQSTM1 accumulation in Lamp2 heterozygous null mice with progressively longer periods of fasting, as compared with nonfasted littermate wild-type female controls. ACTA1 was employed as loading control. (D and E) Representative immunoblots (D) and quantification of ratio of LC3-II (E, top) and SQSTM1 (E, bottom) in cardiac extracts from Lamp2 heterozygous null mice and female littermate wild-type controls subjected to 24 h fasting or provide ad libitum access to diet and treated for CQ or diluent (for 4 h prior to sacrifice). N = 3 /group. P values are by post-hoc test after one-way ANOVA. (F) Representative immunoblots depicting LC3 and SQSTM1 in Lamp2 heterozygous null mice and female littermate wild-type mice subjected to intermittent fasting for 6 wk, and treated for CQ or diluent (for 4 h prior to sacrifice) on a fed day. (G) Representative transmission electron microscopy images of cardiac tissues from Lamp2 heterozygous null mice and female littermate wild-type mice subjected to intermittent fasting, or provided ad libitum access to food for 6 wk, on a fed day. N = 3 /group. Arrows indicate mitochondria with loss of cristal architecture, and arrowheads point to autophagic structures.