Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition
Figure 6
Niclosamide but not rapamycin affects the formation of large ubiquitin-containing aggregates and p62 levels.
(A) A representative GFP-ubiquitin SH-SY5Y cell treated with 20 nM rapamycin for 8 h, prior to PFA fixation and Hoechst staining. GFP signal intensity (vertical axis) for the indicated 5 µm cross-section (white bar) is shown on the right side. (B) SH-SY5Y cells were treated as indicated (5 µM MG132, 10 µM niclosamide, 20 and 100 nM rapamycin) for 8 h, prior to lysis in RIPA buffer. Equal amounts of proteins were separated by 4–20% SDS-PAGE followed by immunoblotting with ubiquitin, SK6 and anti-α-tubulin antibodies. (C) Signal intensities of the GFP-ubiquitin aggregates were measured in three independent wells for a each condition using the automated high-content fluorescence imager. Cells were treated with rapamycin (10, 30, 100 nM) alone (light grey), and together with 5 µM MG132 (dark grey) for 8 h. (D) Same as B but with anti-p62 and anti-α-tubulin antibodies. (E). SH-SY5Y cells were pre-treated with 1 µM epoxomicin for 4 h, prior to cell wash. Mock-DMSO or niclosamide treated cells were then further incubated for the indicated times and processed as in D.