Figure 4

Celastrol blocks protein degradation and promotes accumulation of poly-ubiquinated substrates in human glioblastoma cells. (a) U251N cells were treated with serum-containing and serum-deprived media in the presence and absence of 500 nM 17-AAG (Hsp90 inhibitor), 10 μM MG-132 (proteasome inhibitor) and various concentrations of celastrol for 24 h. Total cell lysates were collected and immunoblotted for ubiquitin. (b and c) Cells were treated with serum-containing and serum-deprived media in the presence and absence of 10 μM MG-132 and 3 μM celastrol for 3–24 h. Total cell lysates were collected and immunoblotted for LC3B (a) and p62 (b). Actin is used as a control for protein loading. (d) In situ immunolabeling of p62 in U251N cells reveals accumulation of aggresomes, as indicated by white arrowheads. Cel, Celastrol 3 μM for 8 h; Rap, Rapamycin 200 nM for 24 h. Scale bar is representative for all four panels. (e) U251N cells were treated with increasing concentrations of celastrol for 24 h and analyzed for lysosomal content as described in the Materials and Methods section. Lysosome number was normalized to untreated cells (100%). Significant differences relative to untreated cells are denoted. (f) U251N cells labeled with LysoTracker Red DND-99 are depicted with cytoplasmic membrane outlines highlighted to clarify cell delineations. Scale bar in the first panel (50 μM) is representative for all the four panels shown. (g) U251N cells were treated with serum-deprived media with or without 50 μM CQ (inhibitor of lysosomal acidification) or 5 mM 3MA (inhibitor of the class III P13K, Vps34) for 1 h after which celastrol was added at various concentrations (0.5–10 μM) for another 24 h. Cell viability was assessed using the MTT assay. No significant differences were observed with CQ or 3MA relative to celastrol alone (LD50: 3.66±0.31 μM with serum deprivation versus 3.50±0.30 μM with CQ and 3.69±0.24 μM with 3MA; P=2.728). (h) U251N cells were treated with serum-deprived media with or without 1 μg/ml CHX, an inhibitor of protein translation, for 1 h, after which celastrol was added at various concentrations (0.3–10 μM) for another 24 h. Cell viability was assessed using the MTT assay. A significant difference in the response to celastrol was noted with CHX-treated cells relative to treatment with celastrol alone (LD50: 5.01±0.24 μM without CHX versus 14.8±88.3 μM with CHX; P=4.38 × 10⁻⁴) (see Statistical analysis section in Materials and Methods). Average values and S.Ds. are reported for triplicate measurements (N=3). Results are representative of at least three independent experiments. **P<0.01, ***P<0.001