Figure 5

mTOR inhibitors block NIM811-induced vacuolization. (a and b) U251 cells were treated with 10 μM NIM811 in combination with 100 μM chloroquine (CQ) or 100 nM bafilomycin A1 (Baf-A) for 24 h. Scale bar=50 μm. (c) Four-hour pretreatment with 100 nM rapamycin or 150 nM torin-2 blocked vacuolization in U251 cells at 48 h. Scale bar=50 μm. (d and e) Western blotting of NIM81-treated cells demonstrated transient stimulation of mTOR as well as AKT phosphorylation at 2-6 h. Bip remained upregulated after 24 h. (f) Cell lysates were collected from 2 h to 10 h of 10 μM NIM811-treated U251 cells, and analyzed for protein ubiquitination