Fig. 2.

Build-up of DRiPs appears to trigger aggresome formation. (A) Inhibition of proteasome and active translation are necessary only for a short time to trigger aggresome formation in MCF10A cells stably expressing Syn–GFP. Thirty minutes after the addition of the indicated inhibitors, samples were vigorously washed and left in a regular medium for additional 3.5 hours. Efficiency of aggresome formation was evaluated in all samples 4 hours after the addition of MG132. (B) MG132 at low concentration demonstrates synergy with puromycin in induction of aggresome formation. MCF10A cells stably expressing Syn–GFP were incubated with indicated concentrations of MG132 with or without 7.35 μM puromycin for 1.5 hours and the fraction of cells with the aggresome was counted. (C) Lower concentrations of puromycin are more efficient in boosting the aggresome formation. HeLa cells stably expressing Syn–GFP were incubated for 3.5 hours with indicated concentrations of MG132 and puromycin (2, 4 or 8 μg/ml correspond to 3.67, 7.35 μM or 14.7 μM, respectively) and the fraction of cells with an aggresome were counted. (D) Canavanine promotes aggresome formation. MCF10A cells stably expressing Syn–GFP were incubated with or without MG132 and canavanine for 2 hours and the fraction of cells with an aggresome were counted. Of note, the kinetics of the aggresome formation induced by low concentrations of MG132 was slower.