Edited by Herman N. Eisen, Massachusetts Institute of Technology, Cambridge, MA, and approved November 20, 2000 (received for review March 24, 2000)
Effect of proteasome inhibitors on antigen processing in EL4 and EL4ad cells. Cells were treated with proteasome inhibitors and then infected with PR8 influenza virus. TCD8+ cells specific for influenza NP366–374 were added 1 h after infection. Brefeldin A was added 5 h after infection. After an additional 4 h, cells were harvested, stained for CD8 and intracellular IFN-γ, and analyzed by cytofluorography. (A) The percentage of CD8+ cells positive for intracellular IFN-γ is represented graphically. (B) To ensure that proteasome inhibitors were not inhibiting IFN-γ production in TCD8+ cells, the assay described in A was performed with the use of EL4 cells titrated with limiting quantities of NP366–374 peptide as antigen-presenting cells and TCD8+ cells exposed to the same inhibitor concentrations as in A. Legend symbols are defined in A.
Effect of proteasome inhibitors on the accumulation of polyUb proteins in EL4 and EL4ad cells. (A and B) EL4 (A) and EL4ad (B) cells treated with inhibitors for 2, 4, and 8 h were analyzed by Western blotting with the use of the FK2 mAb, the binding of which was visualized by chemiluminescence. The inhibitors used were 10 μM lactacystin (LC), 10 μM zLLL (zLLL), 10 μM AAF-cmk (AAF), no inhibitor (NI), 50 μM NLVS (NLVS), and 1 μM epoxomicin (Epox). (C) The increase in FK2 staining was quantitated and is represented graphically.
Effect of proteasome inhibitors on the accumulation of p53 in EL4 and EL4ad cells. Aliquots from cells analyzed in Fig. 2 were Western blotted with the use of polyclonal p53-specific antibodies. The inhibitors used were 10 μM lactacystin (LC), 10 μM zLLL (zLLL), 10 μM AAF-cmk (AAF), 1 μM epoxomicin (Epox), 1 μM boro-LLL (Boro), 1 μM PS-341 (PS-341), and no inhibitor (NI). (A) Antibody binding was visualized by chemiluminescence. (B) The increase in p53 staining was quantitated and is represented graphically.
Effect of AAF-cmk on AAF-amc hydrolysis in EL4ad cells. EL4ad cells were treated with 10 μM AAF-cmk or 10 μM lactacystin for 90 min at 37°C and then incubated with 100 μM AAF-amc. Hydrolysis of AAF-amc was determined by measuring fluorescence at 5–10-min intervals.
Inhibitor concentrations resulting in 80% growth inhibition of EL4 and EL4ad cells
| Inhibitor | EL4, μM | Fold difference inhibitor concentration | |
|---|---|---|---|
| EL4ad + 50 μM NLVS | EL4ad | ||
| Lactacystin | 4 | 0.75 × | 1.25 × |
| zLLL | 0.4 | 1 × | 3.75 × |
| NLVS | 8 | >6 × | >6 × |
| Epoxomicin | 0.03 | 23 × | 50 × |
| Boro-LLL | 0.04 | 12.5 × | >25 × |
| PS-341 | 0.02 | 1 × | 2 × |
| YU101 | 0.25 | 4 × | 8 × |
| AAF-cmk (Sigma) | 20 | 1 × | 1.25 × |
| AAF-cmk (Bachem) | 20 | 1.5 × | 1.5 × |
Cells were cultured in RP10 with varying concentrations of the indicated inhibitors, and the number of viable cells was determined by trypan blue exclusion every 24 h. After 72 h in culture, the concentration of inhibitor necessary to cause an 80% reduction in the number of viable cells relative to control cultures was determined. The fold difference in inhibitor concentration for EL4ad relative to EL4 cells is indicated.
