Figure 3

NIM811-induced vacuolization is sensitive to protein synthesis inhibition. (a) U251 cells were incubated with 10 μM NIM811 containing media for 9 h, then media were replaced with fresh DMEM (10% FBS). Cells were then tracked by live microscopy (scale bar=25 μm) for 24 h. (b) U251 cells were pretreated with 20 μM cycloheximide (CHX) or 10 μg/ml of blasticidin or 20 μM of U0126 respectively for 2 h followed by 24 h 10 μM NIM811 incubation (scale bar=25 μm). (c) Twenty hours of NIM811 treatment leads to accumulation of ubiquitinated proteins, pretreatment with 20 μM cycloheximide for 2 h prevented this build-up. (d) After 24 h or 48 h of 10 μM NIM811 treatment, cells were collected for 20S proteasome activity assay. Cells treated with 1 or 5 μM MG132 served as positive control for this assay