Figure 2.

Failure of Amino Acid Homeostasis Causes Cell Death following Proteasome Inhibition

(A) Viability of NIH 3T3 cells, assessed by the reduction of WST-8 into formazan, after 2 days of growth, following treatment with 10 μM MG-132 for 8 hr and a 20 hr washout in regular medium, with or without cysteine (Cys) and/or asparagine (Asn). Data are means ± SD (n = 4). ^∗p ≤ 0.01, ^∗∗p ≤ 0.001, and ^∗∗∗p ≤ 0.0001. n.s., not significant.

(B) Polyubiquitin (Ubⁿ) and tubulin immunoblots of lysates from NIH 3T3 cells exposed to 10 μM MG-132 treatment for 6 hr, followed by a washout in regular medium, with or without 1 mM Cys supplementation, for the indicated time.

(C) Fluorescence of proteasome reporter-substrates Ub^G76V-GFP (Dantuma et al., 2000), ZsGreen-ODC (Hoyt et al., 2005), and GFP-CL1 (Gilon et al., 1998; Bence et al., 2001) in NIH 3T3 or 293T cells, following treatment with 10 μM MG-132 and a washout for the indicated time, with or without Cys. Data are means ± SD (n = 3). ^∗p ≤ 0.01, ^∗∗p ≤ 0.001, and ^∗∗∗p ≤ 0.0001. n.s., not significant.

(D) Fluorescence of proteasome reporter-substrates ZsGreen-ODC (Hoyt et al., 2005) following treatment with 10 μM MG-132 either alone or together with 1 mM Cys where indicated.

(E) Polyubiquitin (Ubⁿ), tubulin, and NF-κB (p105 and p50) immunoblots of lysates of 293T cells overexpressing p105 and treated with 10 μM MG-132 for 8 hr, either alone or together with 1 mM Cys, where indicated. Representative results of at least three independent experiments are shown.

See also Figure S2.