Figure 4.

Amino Acid Supplementation Suppresses Autophagy Resulting from Proteasome Inhibition

(A) Confocal micrographs of HeLa cells stably expressing GFP-LC3 (Thurston et al., 2009) either untreated or following 10 μM MG-132 treatment, with or without 1 mM Cys, where indicated. Nuclei were stained with H33258.

(B) Quantification of GFP-LC3 dots in at least 100 cells exposed to the indicated treatment. Data are means ± SEM. ^∗∗∗p ≤ 0.0001.

(C) LC3 and tubulin immunoblots of lysates from cells treated with 100 nM Bafilomycin A1 (Baf A1) for 15 hr, or 10 μM MG-132 alone or together with 1 mM Cys for the indicated time.

(D) Quantification of the LC3-II/tubulin ratio in three independent experiments. Values were normalized to untreated cells. Data are means ± SEM. ^∗p ≤ 0.05.

(E) Viability of wild-type and Atg5−/− mouse embryonic fibroblasts, assessed by the reduction of WST-8 into formazan, after 2 days of growth, following treatment with 10 μM MG-132 for the indicated time and a washout in regular medium. Data are means ± SD (n = 4), normalized to cell number. ^∗∗p ≤ 0.001. n.s., not significant.

See also Figure S4.