Figure 4.
ULK-101 Reduces Autophagic Turnover
(A) U2OS cells were treated for 3 hr with the indicated concentrations of ULK-101, with (+) or without (−) 100 nM BafA1 for the final 1.5 hr. Lysates were probed for LC3B and β-actin as a loading control and imaged. A representative from 3 biological replicates is shown.
(B) Relative LC3B-II signal was determined by dividing the LC3B-II signal by the β-actin signal. The difference between (+) and (−) BafA1 bands was calculated for each ULK-101 concentration, and this value was plotted (normalized to 1.0 for the vehicle control). Symbols represent the mean of 3 biological experiments ± SEM. The red line is an EC50 curve fit by non-linear regression.
(C) EGFP-LC3B (green) was imaged in U2OS cells treated for 3 hr with vehicle or 5 μM ULK-101 with (+) or without (−) 100 nM BafA1 added for the final 1.5 hr. Green, EGFP-LC3B; blue, Hoechst-33342 (nuclei). Right panels are 5× magnifications of insets (boxes).
(D) The number of EGFP-LC3B puncta per cell was quantified from images in (C) (≥70 cells per condition). Data are represented as mean ± SEM. Two-tailed, unpaired t test; ***p < 0.001.
(E) U2OS cells were treated with vehicle control (gray bars) or 100 nM AZD8055 (blue bars) supplemented with or without 5 μM ULK-101 for 3 hr. Vehicle control or 100 nM BafA1 was added for the last 1.5 hr, and the relative amount of BafA1-induced LC3B-II (normalized to β-actin) was quantified as in (B). Data are represented as mean of 3 biological replicates ±SEM. Two-tailed, unpaired t test; **p < 0.01.
(F) U2OS cells were treated with full growth media or an Hank's balanced salt solution (HBSS)-based starvation media for 3 hr. ULK-101 was added at the indicated concentrations with (+) or without (−) 100 nM BafA1 for the final 1.5 hr. Lysates were probed for LC3B and β-actin as a loading control and imaged.