KLF15 regulation of autophagy.

<p><b>A</b>. <b>Autophagy marker expression in WT and <i>KLF15</i>^<i>-/-</i> primary hepatocytes and liver tissue</b>. (Top) Hepatocytes were isolated from standard chow-fed 4-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice. Protein lysates were subjected to immunoblotting with the antibodies shown. (Bottom) 2-month-old WT and <i>KLF15</i>^<i>-/-</i> male mice were placed on a HFD (60% kcal from fat) for 8 weeks. Mice were fasted for 5h, then i.p. injected with 10mU/g insulin and sacrificed 10 minutes post-injection. Lysates from flash frozen livers were subjected to immunoblotting with the antibodies shown (n=3). Quantitation graphs are shown next to immunoblots (white bar = WT, black bar = KLF15^-/-). <b>B</b>. <b>Ultrastructural examination of autophagic vesicles in liver tissue from HFD-fed WT and <i>KLF15</i>^<i>-/-</i> mice</b>. WT and <i>KLF15</i>^<i>-/-</i> female mice (n=3) received HFD (60% kcal from fat) for 4 weeks starting at age 3 months. Mice were fasted for 21h before sacrifice and liver tissue was removed and prepared for transmission electron microscopy (TEM) analysis as described in Materials and Methods. (Top) Electron micrographs (original magnification = 43,000x) of liver sections showing clustering of autophagic vesicles (indicated by red arrows) in <i>KLF15</i>^<i>-/-</i> versus WT hepatocytes. Scale bar = 200nm. (Bottom) Quantitation of autophagic vesicles in WT and <i>KLF15</i>^<i>-/-</i> liver. TEM was used to visualize autophagic vesicles in intact hepatocytes. Values = number of autophagic vesicles per cell calculated from 15 hepatocytes per group. <b>C</b>. <b>Chloroquine treatment of primary hepatocytes</b>. Hepatocytes were isolated from 3.5-month old male WT and <i>KLF15</i>^<i>-/-</i> mice and treated with vehicle or 50µM chloroquine (CQ) for 2h prior to harvest. Protein lysates were subjected to immunoblotting with an antibody against LC3B. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g006" target="_blank">Figure 6</a> primary hepatocyte experiments were performed at least twice in triplicate; each lane indicates a technical replicate. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g006" target="_blank">Figure 6A</a> liver blot, each lane represents one mouse. Statistical analysis was performed using Student’s t-test for unpaired samples. Values = mean ± SEM. *p<0.05; **p<0.01.