Figure 3.
Effect of proteasome inhibition is enhanced in older cultures, is stable over time, and does not depend on protein synthesis. A, Clasto-lactacystin β-lactone was applied for various lengths of time and the average dye uptake is plotted over time as mean ± SEM (0 min, 41.7 ± 1.4 a.u., n = 21; 15 min, 44.4 ± 4.5 a.u., n = 13; 60 min, 66.8 ± 5.2 a.u., n = 13; 120 min, 62.9 ± 3.2 a.u., n = 25; 300 min, 70.31 ± 2.8 a.u., n = 4). B, FM4–64 dye uptake as a function of days in culture and plotted as mean ± SEM (day 12 Ctrl, 48.6 ± 7.3 a.u., n = 3; Clast, 57.4 a.u., n = 1; day 13 Ctrl, 55.5 ± 4.6 a.u., n = 18; Clast, 75.0 ± 9.5 a.u., n = 9; day 14 Ctrl, 58.1 ± 5.5 a.u., n = 8; Clast, 81.0 ± 8.3 a.u., n = 6; day 15 Ctrl, 65.2 ± 4.4 a.u., n = 7; Clast, 75.4 ± 7.4 a.u., n = 7; day 16 Ctrl, 65.6 ± 3.3 a.u., n = 12; Clast, 97.8 ± 8.6 a.u., n = 12; day 17 Ctrl, 71.7 ± 11.9 a.u., n = 7; Clast, 98.4 ± 8.3 a.u., n = 9; day 18 Ctrl, 68.6 ± 3.1 a.u., n = 13; Clast, 112.1 ± 10.9 a.u., n = 9; day 19 Ctrl, 59.8 ± 4.2 a.u., n = 15; Clast, n = 0, day 20 Ctrl, 67.9 ± 6.7 a.u., n = 8; Clast, 110.1 ± 6.2 a.u., n = 20; day 21 Ctrl, 62.8 ± 9.1 a.u., n = 6; Clast, 130.4 ± 6.0 a.u., n = 9). Controls (open circles) and 2 h proteasome inhibitor-treated cultures (filled triangles) were analyzed. C, D, The 2 h increase in the vesicle recycling pool is not dependent on protein synthesis. C, Cultures were treated with vehicle (1% DMSO) or the protein synthesis inhibitor cycloheximide (Cyclo) (100 μm) for 2 h in the absence or presence of clasto-lactacystin β-lactone (10 μm). Average dye uptake is plotted as mean ± SEM (Ctrl, 46.56 ± 1.97; Clast, 64.45 ± 3.26; Cyclo, 52.02 ± 3.22; Cyclo/Clast, 62.81 ± 3.93, n = 9 in all conditions). Two-way ANOVA followed by Tukey's post hoc analysis at p < 0.05 revealed a significant difference in clasto-lactacystin β-lactone treated cultures (*relative to Ctrl) with no effect of cycloheximide. D, Cultures were also treated with the irreversible protein synthesis inhibitor emetine (100 μm) for 2 h in the absence or presence of clasto-lactacystin β-lactone (10 μm). Average dye uptake is plotted as mean ± SEM (Ctrl, 41.03 ± 2.13; Clast, 56.12 ± 2.02; Emetine, 39.5 ± 2.56; Emetine/Clast, 50.54 ± 3.35, n = 9 in all conditions). Two-way ANOVA followed by Tukey's post hoc analysis at p < 0.05 again revealed a significant difference in clasto-lactacystin β-lactone treated cultures (*relative to Ctrl) with no effect of emetine.