Differentiated C2.7 cells carrying control or TRIM32-targeting shRNA were incubated with 400 μM dexamethasone for 4 hours. Cells were fixed before and after dexamethasone treatment by direct addition of 5% glutaraldehyde (Merck Millipore, 1042390250) and 4% paraformaldehyde (Sigma-Aldrich, 441244) in 0.1 M cacodylate buffer (pH 7.4; Sigma-Aldrich, 20840-100G-F) to the culture medium. After a 20-min incubation at room temperature, the fixation solution was replaced by 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), and fixation was prolonged overnight. Cells were then embedded in EPON resin [for 25 ml, 12 g of glycid ether (SERVA, 21045.02), 8 g of 2-dodecenysuccinic acid anhydride (SERVA, 20755.02), 5 g of methylnadic anhydride (SERVA, 29452.02), and 560 μl of N-benzyldimethylamine (Electron Miscroscopy Sciences, 11400-25)]. Subsequently, 55-nm sections were cut and stained with uranyl acetate and lead citrate. Cell sections were examined using an 80-kV CM100 transmission electron microscope (Phillips). Three different grids with sections obtained from the same preparations were statistically evaluated. For every grid, the average number of degradative compartments (amphisomes, autolysosomes, and lysosomes) per cell section was determined by counting 25 randomly selected cell profiles.

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