Figure 1.

Pharmacological inhibition of macroautophagy up-regulates CMA. NIH3T3 mouse fibroblasts were maintained in culture in the presence (+ serum) or absence of serum (− serum) without additions (None) or in media supplemented with 10 mM 3-MA. After 6 h, lysosomes were isolated as described under Materials and Methods. (A) CMA activity. Intact isolated lysosomes (left) or lysosomes disrupted by hypotonic shock (right) were incubated in an isotonic medium or in water, respectively, with a ¹⁴C-labeled pool of cytosolic proteins for 30 min at 37°C. Reactions were stopped by addition of TCA, and proteolysis was calculated as the percentage of acid precipitable radioactivity transformed to acid soluble at the end of the incubation. Values are the mean + SE of three different experiments with triplicate samples (**p < 0.001, differences with untreated cells; ^§p < 0.01, differences with serum supplemented cells). (B) Lysosomal uptake of CMA substrates. The isolated lysosomes, pretreated or not with a cocktail of protease inhibitors, were incubated with 10 μg of GAPDH for 20 min at 37°C. At the end of the incubation, lysosomes were collected by centrifugation and the amount of GAPDH taken up by lysosomes was calculated as described under Materials and Methods. Inset shows a representative immunoblot of the association of GAPDH to lysosomes (lysosomes treated with protease inhibitors). Values are expressed as percentage of the total amount of GAPDH added into the incubation media and are mean + SE of four different experiments. (C) Levels of CMA components. Total cellular homogenates and lysosomes (50 μg of protein) were subjected to SDS-PAGE and immunoblot for the indicated proteins. The densitometric quantification of two immunoblots from different experiments is shown on the right. Values are mean + SE, and they are expressed as -fold increase the value in untreated cells maintained in media supplemented with serum, which was given an arbitrary value of 1. Actin is shown as a loading control in homogenates.