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Results: 7

1.
FIGURE 3.

FIGURE 3. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Confocal images of GFP-labeled Htt fragments expressed in either weak [PSI+]/[PIN+] or strong [PSI+]/[PIN+] yeast. Images were obtained 26 h after induction of Htt expression. The left panel shows the GFP fluorescence, while the right panel shows an overlay of GFP fluorescence, PI staining, and DIC images of the yeast.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
2.
FIGURE 6.

FIGURE 6. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Rescue of Htt toxicity by constitutive expression of MC in weak and strong [PSI+]/Δrnq1 yeast. A, spot assays showing the effect of MC on Htt toxicity in weak and strong [PSI+]/Δrnq1 yeast. Yeast containing HttQ103, HttQP103, or empty Ura3 vector were transformed with the empty Leu2 vector or the MC vector. Yeast were spotted on SGal-Ura-Leu plate and grown for 6 days at 30 °C. The spot assays comparing the growth of yeast in the presence and absence of MC were done on the same SGal-selection plate. B, growth curves of weak and strong [PSI+]/Δrnq1 yeast constitutively expressing MC and either HttQ103 or HttQP103. The control is [PSI+]/Δrnq1 yeast constitutively expressing MC with the empty vector.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
3.
FIGURE 2.

FIGURE 2. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Effect of Htt fragments expression on the growth of weak and strong [PSI+]/[PIN+] yeast. Yeast without prion [psi]/[pin] were used as control. A, spot assay of yeast containing either empty vector plasmid or plasmid expressing the following Htt fragments: HttQ25, HttQP25, HttQ103, and HttQP103. Yeast were grown on SGal-Ura plate for 6 days at 30 °C. B, growth of yeast variants with empty vector or expressing different Htt fragments. Yeast grown in SGal-Ura liquid culture were plated on SD-Ura plates at indicated time points. The number of colonies on each plated was counted after 4 days at 30 °C.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
4.
FIGURE 7.

FIGURE 7. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Expression of HttQ103 or HttQP103 in [psi]/[PIN+] yeast. A, spot assay of [psi]/[pin] and [psi]/[PIN+] yeast containing empty vector or expressing either HttQ103 or HttQP103. The spot assays comparing the growth of [psi]/[pin] and [psi]/[PIN+] yeast were done on the same SGal-Ura plate. B, growth curves of [psi]/[PIN+] yeast with empty vector or expressing either HttQ103 or HttQP103. Yeast were grown in SGal-Ura liquid culture and plated on SD-Ura plates at indicated time points. Colony count from each plate following incubation at 30 °C for 4 days was used to measure yeast growth. The number of colonies on each plated was counted after 4 days at 30 °C. C, confocal images of [psi]/[PIN+] yeast expressing HttQ103 or HttQP103. Yeast were incubated in galactose for 26 h to express Htt fragments. The left panel shows the GFP fluorescence, while the right panel shows an overlay of GFP fluorescence, PI staining, and DIC images of the yeast.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
5.
FIGURE 5.

FIGURE 5. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Effect of Htt fragments expression on the growth of weak and strong [PSI+]/Δrnq1 yeast. A, spot assays comparing Htt toxicity of [PSI+] yeast in the presence and absence of [PIN+]. The comparison between the growth of the [PSI+]/[PIN+] and the [PSI+]/Δrnq1 yeast were done on the same SGal-Ura selection plate. B, growth curves of weak and strong [PSI+]/Δrnq1 yeast with empty vector or expressing different Htt fragments. Yeast were grown in SGal-Ura liquid culture and plated on SD-Ura plates at indicated time points. The number of colonies on each plated was counted after 4 days at 30 °C. The red lines are from the data in Fig. 2B. C, confocal images of weak and strong [PSI+]/Δrnq1 yeast expressing HttQ103 or HttQP103. Yeast were incubated in galactose for 26 h to express Htt fragments. The left panel shows the GFP fluorescence, while the right panel shows an overlay of GFP fluorescence, PI staining, and DIC images of the yeast.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
6.
FIGURE 4.

FIGURE 4. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Rescue of Htt toxicity by constitutive expression of the MC fragment of Sup35. A, spot assay of weak and strong [PSI+]/[PIN+] yeast expressing Htt fragments and the MC fragment of Sup35. Yeast containing HttQ103, HttQP103, or empty Ura3 vector were transformed with the empty Leu2 vector or the MC vector. The yeast were spotted on SGal-Ura-Leu plates and grown for 6 days at 30 °C. The spot assays were done on the same SGal-Ura-Leu plate. B, effect of MC on the growth of weak and strong [PSI+]/[PIN+] expressing HttQ103 or HttQP103. Yeast containing empty vector or the Htt vectors were transformed with a plasmid that constitutively expresses MC. Yeast were grown in SGal-Ura-leu liquid culture and plated on SD-Ura-Leu plates at indicated time points. The number of colonies on each plated was counted after 4 days at 30 °C. The red lines are from the data in Fig. 2B. C, Western blot of MC level in yeast expressing Htt fragments. Lysates were prepared from strong [PSI+]/[PIN+] yeast expressing MC in addition to HttQ103, HttQP103, or empty Ura3 vector, which were grown in SGal-Ura-Leu medium for 24 h. Yeast total lysates and supernatant of high speed centrifugation were probed with an anti-Sup35 antibody detecting both MC fragment and wild type Sup35. Pgk1 protein level was used as a loading control. D, spot assay of weak and strong [PSI+]/[PIN+] yeast expressing Htt fragments and Sup45. Yeast containing HttQ103, HttQP103, or empty Ura3 vector were transformed with the empty Trp1 vector or Sup45 vector. The yeast were spotted on SGal-Ura-Trp plate and grown for 6 days at 30 °C. The spot assays comparing the growth of Sup45 expressing and empty vector yeast were done on the same SGal-Ura-Trp plate.

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.
7.
FIGURE 1.

FIGURE 1. From: Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast.

Characterization of weak and strong [PSI+] variants. A, curing of the weak (w) and strong (s) [PSI+] yeast variants by guanidine inactivation of Hsp104. Yeast were grown in SD with 5 mm guanidine and at the indicated generations, the extent of curing was determined by plating on 1/2YPD plates (0.5% yeast extract, 2% peptone, and 2% glucose, 2% agar). Colonies from each plate were counted after incubation at 30 °C for 4 days. [PSI+] cells yield white colonies on 1/2YPD plates, while cured cells ([psi]) yield red colonies. The data are a compilation from three independent experiments. B, Western blot of the total and soluble Sup35 in yeast lysates. Yeast lysates were centrifuged at 100,000 rpm for 10 min in the Beckman TL-100 ultracentrifuge and the total lysates (T) and the supernatant fraction (S) were run on SDS gels. The prion phenotypes are as follows: lanes 1 and 2, [psi]; lanes 3 and 4, weak [PSI+]; lanes 5 and 6, strong [PSI+]. Pgk1 was used as an internal loading control. The percent of the total Sup35 in the supernatant is given beneath the Western blot. C, Western blot of Sup35 showing the total Sup35 (T) and the resuspended pellet (P) following high speed centrifugation. The prion phenotypes are as follows lanes 1 and 2, weak [PSI+]; lanes 3 and 4, strong [PSI+].

Xiaohong Zhao, et al. J Biol Chem. 2012 July 6;287(28):23346-23355.

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