Figure 2: Autophagy is increased in HT-29 cells by serum deprivation and inhibited by 3-methyladenine (3-MA) and Atg7 siRNA. (a) Cells contain basal levels of cytosolic vacuoles in serum-containing media. Cytosolic vacuoles (indicated with arrows) dramatically increased at 24 hours of incubation in serum-free media. (b) Western blots showing silencing of Atg7 by siRNA, but not control RNA (ctrl) relative to mock transfection, conversion of LC3β I into LC3β II in vehicle and control RNA-treated cells over time, and inhibition of LC3β II conversion in 3-MA (10 mM) and Atg7 siRNA (10 ng) treated cells over time in serum-free media. Beta-actin blots show equal protein loading in all samples. (c) Graph of protein quantitations from western blots showing degradation of p62 in vehicle and control RNA treated cells over time, and inhibition of p62 degradation in 3-MA (10 mM) and Atg7 siRNA (10 ng) treated cells over time in serum-free media. One representative western blot out of 3 utilized for the p62 quantitation is shown. *Indicates significantly lower p62 levels relative to 0 hours, .