Figure 3. Niclosamide induces formation of ubiquitinated Tax protein aggregate.

A) HeLa cells were co-transfected with Tax-GFP and Ubiquitin-HA, and the transfected cells were treated with DMSO, niclosamide (10 μM) or MG-132 (5 μM). 45 minutes after treatment, cells were stained with anti-HA and examined with fluorescence imaging. B) Upper panel: HEK293 cells were co-transfected with Tax and ubiquitin-HA. Transfected cells were treated with niclosamide for 4 hours, and the total lysates were immunoprecipitated with anti-Tax followed by anti-ubiquitin blot. Lower panel: HEK293 cells were transfected with Tax-GST. Transfected cells were treated with niclosamide for 2 hours, and the insoluble pellets were dissolved in 1% SDS/50mM Tris-Cl (pH7.6) and further diluted into RIPA buffer (the final concentration of SDS is 0.1%). The GST-pulldown was analyzed with anti-ubiquitin blot. C) MT-4 cells were pre-treated with DMSO, MG-132 (5 μM) or chloroquine (CQ, 50 μM), followed niclosamidetreatment. 24 hours later, the cells were lysed with 1% Triton X-100, and the remaining insoluble pellets (P) were dissolved in 1% SDS buffer. The cellular extracts from soluble fractions (S) and insoluble fractions (P) were examined with immunoblot. D) and E) MT-2 and MT-4 cells were treated with niclosamide. Total protein lysates, soluble proteins and insoluble protein extracts were examined with anti-ubiquitin and anit-ubiquitin-K48 immunoblots.