Research Paper Volume 5, Issue 11 pp 802—812

Figure 5. Overproducing Hsp104 mitigates aggregate accumulation and restores proteasome function in aged cells. (A) Relative levels of Hsp104 produced from the wt HSP104 and GPD promoters as determined by anti-Hsp104 immuno-blot analysis (n=2). (B) Percentage of cells with Ssa2-GFP foci following heat stress in the wt and Hsp104 overproducing (Hsp104↑) strains. Time point “0” represents cells after 30 min incubation at 42°C, whereas subsequent time points represent cells following the indicated time of recovery at 30°C (n=2). (C) The effect of Hsp104 overproduction on aggregate formation. Representative image of Ssa2-GFP and ΔssCPY*-GFP in young and aged, wt and Hsp104 overproducing cells. (D) Percentage of aged wt and Hsp104 overproduction cells with Ssa2-GFP or ΔssCPY*-GFP foci (n=2). (E) Relative half-life of β-gal in wt and Hsp104 overproducing young and aged cells. Values were normalized to the half-life in wt young cells (n=3). (F) Relative levels of Rpt1p (19S subunit), 20S core proteins, and proteasome specific activity (rate of hydrolysis of suc-LLVY-AMC) in Hsp104 overproducing cells compared to wt cells. (n≥2). (G) Relative levels of soluble β-gal in wt and Hsp104 overproducing cells normalized to total protein (see Experimental procedures for details) (n=2). Error bars represent standard deviation. For statistical analysis, the paired two-tailed t-test was used where *P<0.05, **P<0.01, ***P<0.001 and n.s = no significant difference. (Scale-bar represents 10μM). (H) Life-span curves of wt and Hsp104 overproducing cells. Mean replicative life-spans are: wt (28 ± 0), Hsp104 overproduction (29.5 ± 1.5) (n=2). (n= sets of analysis).