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1.
Figure 6.

Figure 6. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Genetic interactions between Slt2p, Mca1p, and Cdk8p. Wild-type (RSY10), mca1Δ (RSY1046), slt2Δ (RSY1057), mca1Δ slt2Δ (RSY1023), and cdk8Δ slt2Δ (RSY1021) midlog cultures were harvested and tested for sensitivity to H2O2 as described in Figure 3.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
2.
Figure 3.

Figure 3. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Cyclin C stability regulates oxidative stress-induced cell death. (A) Log-phase cultures of the indicated genotype were serially diluted (1:10) and plated onto complete minimal medium without (control) or with (right panels) H2O2 as indicated. Strains used were (top to bottom) RSY10, RSY391, RSY1057, and RSY1007. Plates were incubated at 23° for 3 days and then photographed. (B) Wild-type strain RSY10 was transformed with either the myc–cyclin C (pLR101) or the myc–cyclin CA110V (pLR102)-expressing plasmids. The cultures were grown to midlog phase and treated as described in A except that the minimal medium used selected for plasmid maintenance.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
3.
Figure 4.

Figure 4. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Pkc1p-directed MAP kinase cascade targets cyclin C for destruction through a novel destruction element. (A) Wild-type strain RSY10 was transformed with plasmids expressing myc–cyclin CA110V and either the wild-type (BCK1) or activated (BCK1-20) allele of the MEK kinase. Cultures were harvested at midlog phase in the absence of stress. myc–cyclin CA110V levels were determined as in Figure 1. Tub1p serves as the loading control. Mab, myc monoclonal antibody used during immunoprecipitations, which is cross reactive with the secondary antibody used to develop the myc–cyclin C signal. (B) A space-filling model of cyclin C (white) and Cdk8p (shaded) is presented (see materials and methods for details). The arrows indicate the predicted locations of residues Ala110 and Glu170.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
4.
Figure 5.

Figure 5. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Slt2p and cyclin C regulate programmed cell death in opposite ways. (A) A wild-type culture (RSY10) following exposure to 0.4 mm H2O2 was stained with DAPI to identify the nuclei and probed for the presence of double-strand breaks by TUNEL assays (see materials and methods for details). The merged image is shown on the right. Magnification ×400. (B) The wild-type culture RSY10 before (top, left) and following (top, right) H2O2 treatment (0.4 mm) were examined by TUNEL assays. Isogenic bck1Δ (RSY851) and cycCΔ (RSY391) mutants were also examined by TUNEL assays following H2O2 exposure. (C) Strains lacking SLT2 (RSY1057), cyclin C (RSY391), or both (RSY1007) were assayed for double-strand breaks by TUNEL. The inserts depict higher magnifications of selected cells in the same field to highlight nuclear breakdown.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
5.
Figure 2.

Figure 2. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Slt2p mediates only ROS-induced cyclin C destruction. (A) Wild-type SLT2 (RSY10) and isogenic null mutant slt2Δ (RSY1057) expressing myc–cyclin C were subjected to heat shock (37°) for the times indicated (in minutes). myc–Cyclin C levels were determined as in Figure 1. Tub1p serves as a loading control. The asterisk indicates a nonspecific cross-reacting protein with the myc Mab. (B) The cultures described in A were grown to midlog phase and then exposed to H2O2 treatment (0.4 mm) for the indicated times (in hours) and myc–cyclin C levels were determined as before. (C) The slt2Δ strain (RSY1057) expressing myc–cyclin C was transformed with the stl2K54R kinase dead allele of SLT2 or vector control. An oxidative stress time course was conducted and cyclin C levels were visualized as described in B.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
6.
Figure 1.

Figure 1. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

Oxidative stress-induced destruction of cyclin C requires the Pkc1p MAP kinase cascade. (A) Diagram of the Pkc1p-controlled MAP kinase pathway with corresponding kinase designations on the left. (B) Wild-type BCK1 (RSY10) and bck1Δ mutant (RSY815) strains transformed with the myc–cyclin C expression plasmid were subjected to oxidative stress (0.4 mm) for the times indicated (in hours). myc–cyclin C levels were monitored by Western blots of immunoprecipitates. Tub1p served as a loading control. Molecular weight standards (in kilodaltons) are indicated on the left. (C) myc–cyclin C levels were determined in the wild-type strain RSY10 harboring either the wild-type (BCK1) or the constitutively active (BCK1-20) BCK1 allele on a plasmid. Cultures were harvested in midlog phase in the absence of stress. (D) The experiment described in C was repeated in the mkk1 mkk2 double mutant (RSY921). Tub1p served as a loading control. The asterisk indicates a nonspecific cross-reacting protein with the myc Mab.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.
7.
Figure 7.

Figure 7. From: Regulation of the Oxidative Stress Response Through Slt2p-Dependent Destruction of Cyclin C in Saccharomyces cerevisiae.

The human cyclin C regulates the ROS response in yeast. (A) HA-epitope-tagged allele of the HcycC was subjected to Western blot analysis. The vector lane (Vec) serves as a control for nonspecific cross-reactivity of the HA Mab. Tub1p serves as a loading control. Molecular weight standards (in kilodaltons) are indicated on the left. (B) The aberrant vegetative expression of the meiosis-specific spo13–lacZ reporter gene was analyzed in a cycCΔ mutant strain harboring the vector control, the yeast cyclin C expression plasmid (pLR101), or the human HcycC-expressing plasmid (pKC409). Plate assays indicating lacZ expression by cleavage of 5-bromo-4-chloro-3-indole-β-d-galactoyranosidase (X-gal) of two independent transformants are depicted. (C) H2O2 sensitivity assays were conducted as described in Figure 3 with the strains indicated in B except that the first dilution in the series was spotted onto medium containing different amounts of H2O2 as indicated. The plates were incubated for 3 days at 23° and then photographed.

Elizabeth Krasley, et al. Genetics. 2006 March;172(3):1477-1486.

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