Figure 3 (See previous page).

Molecular phenotype of lipid and misfolded protein response. (A) Immunoblot of Harderian gland samples for the lipid droplet marker PLIN2, the protein aggregate marker SQSTM1, and ubiquitin as a marker for misfolded protein response. Consistently in the absence of autophagy, atg7^−/− gland samples accumulated PLIN2, SQSTM1 protein, and SQSTM1 aggregates (higher molecular weight species of SQSTM1, which is not resolved on the SDS gel), as well as ubiquitin in comparison to the Atg7^f/f control samples. (B) Determination of lipid composition by thin layer chromatography. HaGl extracts, normalized to their wet weight. Four different animals of each genotype were separated by thin layer chromatography with solvents for a broad lipid spectrum. In extracts of atg7^−/− glands the amount of free cholesterol (FC) is increased. A prominent band corresponds to the triglycerides analog (TG) 1-Alkyl-2,3-diacylglycerol, which according to the literature is the most abundant class of lipids of the Harderian gland. Wax esters and cholesterol esters (CE), free fatty acids (FFA), Ceramides (Cer), polar lipids (mostly phospholipids, PL). (C) Quantification of free cholesterol from the TLC. Analysis of the band intensity showed a 30% increase in of free cholesterol compared relative to the gland weight (P = 0.0017). (D) Immunohistochemistry for the protein aggregate marker SQSTM1. While no SQSTM1 is detected in sections of the Atg7^f/f control, the signals for SQSTM1 have a granular and circular appearance and were sometimes localized in a juxtanuclear position or at the base of the duct in sections of atg7^−/− glands. Here the size of the SQSTM1 granule indicates large inclusions and deposits, which were found in a mosaic like pattern affecting cells unequally. In addition, SQSTM1 is weak and diffusely distributed in the cytoplasm and in the secretions within the ductal lumen. The diffuse SQSTM1 distribution may reflect a more immature state of potentially not further aggregated cellular forms of misfolded proteins. Black arrowhead corresponds to large vacuoles found in atg7^−/− cells that did not stain for SQSTM1. L, ductal lumen. Size bar = 20 μm. (E) Ubiquitin accumulation in atg7^−/− glands. Ubiquitinated proteins are highly abundant in the cytoplasm of atg7^−/− ductal cells compared to controls. Ubiquitin staining appears partly granular indicating aggregates. Cell debris containing ubiquitin can be detected in the ductal lumen (L). Size bar = 20 μm. (F) Cholesterol aggregating cells of atg7^−/− glands. Filipin III binds to diffuse and aggregated cholesterol in atg7^−/− glands. Such cells highly accumulating cholesterol (white arrow) were absent in control sections. Some vacuoles exclude cholesterol (white arrowhead). Size bar = 20 μm. (G) Coimmuno-fluorescence of SQSTM1 protein aggregates and PLIN2 lipid droplets. Sections of atg7^−/− HaGls reveal an overall higher abundance of PLIN2-positive lipids droplets. SQSTM1 staining is granular and diffuse. Interestingly, discrete cells accumulate both PLIN2-positive lipid droplets and SQSTM1, while this is not observed in cells of control animals. PLIN2 is stained in red, SQSTM1 in green, nuclei are stained in blue by Hoechst. Size bar 20 μm. (H) Single cell laser scanning image of a SQSTM1 and PLIN2 double-positive cell. PLIN2 staining has a circular, droplet like appearance, while SQSTM1 stains large granules and diffusely in the cytoplasmic. SQSTM1 staining is close to the droplets but does not entirely cover the PLIN2 positive structures. Largely these markers appear separate. Note that in this particular cell the nucleus is heavily deformed. PLIN2 stains in red, SQSTM1 in green, nuclear stain Hoechst in blue. Size bar = 5 μm. (I) Gas chromatographic analysis of neutral lipids of the HaGl. Free cholesterol is markedly increased in lipid extracts (n = 3, P = 0.06), whereas triglycerides and free fatty acids do not differ.