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Results: 4

1.
Figure 1

Figure 1. PNM mutants are distinguished by their effective inhibitory ratios. From: Dominant Prion Mutants Induce Curing Through Pathways That Promote Chaperone-Mediated Disaggregation.

a. Wildtype (HSP104/HSP104) or heterozygous disruption (HSP104/Δ) diploid strains expressing wildtype (WT) and PNM mutants (Q24R, G58D) from P35 at the indicated ratios were spotted on rich (1/4 YPD) or adenine deficient (−ADE) media to analyze the [PSI+] phenotype. Wildtype [PSI+] and [psi] diploids (74-D694) were included as controls. b. To determine the frequency of prion loss, wildtype meiotic progeny (n ≥ 19 for each strain) were isolated from the diploids described in (a), and the percentage of [psi] colonies was determined.

Susanne DiSalvo, et al. Nat Struct Mol Biol. ;18(4):486-492.
2.
Figure 3

Figure 3. PNM mutants alter the accumulation of propagons but not their transmission. From: Dominant Prion Mutants Induce Curing Through Pathways That Promote Chaperone-Mediated Disaggregation.

a. Lysates from haploid wildtype yeast strains expressing Sup35 (WT or mutants) from PtetO2 were analyzed by SDD-AGE and immunoblotting for Sup35. Wildtype [PSI+]Strong, [PSI+]Weak, and [psi] yeast strains are shown as controls. b. The number of propagons present in individual cells was determined for the indicated strains, as described in (a). Horizontal lines on boxes indicate 25th, 50th, and 75th percentiles; error bars indicate 10th and 90th percentiles, and dots represent outliers (n≥39; *p≤ 0.0001 in comparison with WT). c. The proportion of Sup35 transmitted to daughter cells (circles) or to mother cells (squares) was determined by fluorescence loss in photobleaching (FLIP) of a [PSI+] strain expressing Sup35-GFP alone ([PSI+]) or with a second copy of Sup35 (WT-GFP + WT) or G58D (WT-GFP + G58D) from PtetO2. Error bars represent standard error of the mean from three independent experiments, each analyzing at least 10 cells.

Susanne DiSalvo, et al. Nat Struct Mol Biol. ;18(4):486-492.
3.
Figure 2

Figure 2. PNM mutants incorporate into wildtype aggregates and alter multiple events in prion propagation. From: Dominant Prion Mutants Induce Curing Through Pathways That Promote Chaperone-Mediated Disaggregation.

a. HA-tagged Sup35 (WT or mutants) expressed from P35 in haploid [PSI+] (+) or [psi] (−) yeast strains, which also expressed untagged Sup35, was immunoprecipitated with anti-HA serum (Ab) and analyzed by SDS-PAGE and anti-Sup35 immunoblotting. b. [psi] haploids expressing Sup35 (WT or mutants) from P35 and a fluorescent reporter of translation termination efficiency (GST-UGA-DsRed-NLS) were mated to wildtype [PSI+] or [psi] (74-D694) cells, and the percentage of fluorescent zygotes was scored. Error bars represent standard deviation from three independent experiments, each analyzing at least 15 zygotes per cross (*p=0.039 in comparison with WT). c. The fluorescence intensities of zygotes isolated from the indicated crosses as described in (b) were determined. Horizontal lines on boxes indicate 25th, 50th, and 75th percentiles; error bars indicate 10th and 90th percentiles, and dots represent outliers (n≥30; *p=0.0009). d. Lysates from wildtype haploid strains expressing an additional copy of Sup35 (WT or mutants) from PtetO2 were incubated in SDS at the indicated temperatures before SDS-PAGE and quantitative immunoblotting for Sup35 (percentage of Sup35 at the indicated temperatures relative to 100°C). Error bars represent standard deviation (n ≥ 6, *p = 0.0003, **p = 0.0001, ***p=0.008 in comparison with WT at the same temperature).

Susanne DiSalvo, et al. Nat Struct Mol Biol. ;18(4):486-492.
4.
Figure 4

Figure 4. PNM expression promotes Hsp104-mediated disassembly of aggregates. From: Dominant Prion Mutants Induce Curing Through Pathways That Promote Chaperone-Mediated Disaggregation.

a. The number of propagons present in individual cells was determined for diploid [PSI+] strains expressing one endogenous copy of SUP35 and one copy of SUP35 (WT or mutants) from PtetO2 in a wildtype background (HSP104/ HSP104) or in a heterozygous disruption background (HSP104/Δ). Box plots are as described in the legend to Fig. 3b. n≥10 cells per strain; *p < 0.05, in comparison with the corresponding HSP104/HSP104 strain. b. Lysates of diploid strains expressing Sup35 (WT) and PNM mutants in the indicated ratios were analyzed by SDD-AGE and immunoblotting for Sup35. Wildtype (+) and heterozygous disruption of HSP104 (Δ) are indicated. c. Lysates from BSC783/4c and 74-D694 [PSI+] haploids were analyzed by SDS-PAGE and quantitative immunoblotting for Hsp104 and Sup35. Error bars represent standard deviation (n=5, *p=0.0031). d. Haploid [PSI+] cells co-expressing endogenous SUP35 and a second copy of SUP35 (WT or mutants) from PtetO2 were transformed with pHSE-Hsp104 (cenP104), SB590 (2μP104), pLH102 (2μPGPD) or a vector control, and the percentage of [psi] colonies was scored after plasmid loss. Error bars represent standard deviation from three independent experiments, each analyzing a total of 50 colonies (*p<0.05, in comparison with vector control for the same strain). e. Lysates from diploid [PSI+] strains expressing two copies of SUP35 (WT) or one wildtype and one mutant copy of SUP35 from PtetO2 were incubated in SDS at 50°C or 100°C before SDS-PAGE and quantitative immunoblotting for Sup35, and the ratio of signal before and after cycloheximide treatment was determined. Error bars represent standard deviation (n≥3, *p=0.029, **p=0.011).

Susanne DiSalvo, et al. Nat Struct Mol Biol. ;18(4):486-492.

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