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1.
Figure 5

Figure 5. STP1 is required for hypoxic and normoxic sterol homeostasis. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. Outline of C. neoformans ergosterol biosynthetic pathway. Genes encoding Sre1-dependent (bold) and Stp1-dependent (underlined) enzymes are listed. B. Wild-type, stp1Δ and sre1Δ cells were grown for 2 hours at 21% or 3% O2. Total sterols were extracted and analyzed by gas chromatography. Ergosterol and sterol intermediates were quantified and normalized to wild-type 21% oxygen ergosterol levels and plotted. Error bars represent the standard deviation from three biological replicate experiments.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.
2.
Figure 3

Figure 3. STP1 is required for virulence. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. C. neoformans serotype A wild-type and stp1Δ cells were grown in YES medium at 37°C. Cell numbers were determined at one hour intervals for 12 hours. Error bars represent one standard deviation from three biological replicates. B. Female Balb/c mice (n=10/strain) were infected via tail vein injection with the indicated C. neoformans strains and mouse survival was monitored.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.
3.
Figure 4

Figure 4. STP1 is required for both Sre1-dependent and Sre1-independent gene expression under low oxygen. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. Venn diagram comparing statistically-significant genes more highly expressed in wild-type versus sre1Δ and stp1Δ cells under low oxygen. B. Venn diagram comparing statistically-significant genes with reduced expression in wild-type versus sre1Δ and stp1Δ cells under low oxygen. C. Quantitative PCR analysis on genes more highly expressed in wild-type versus sre1Δ or stp1Δ cells. Wild-type, sre1Δ, and stp1Δ cells were grown under ambient (21%) or 3% oxygen for 2 hours, and RNA was harvested. RNA level for the indicated genes was quantified by real time RT-PCR and normalized to that in wild-type cells at 21% oxygen. Error bars represent standard deviation from three biological replicate experiments.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.
4.
Figure 6

Figure 6. Sre1 pathway is required for growth and viability in the presence of sterol synthesis inhibitors. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. Wild-type, sre1Δ and stp1Δ cells were grown for 48 hours in liquid YES medium. Final cell density was determined using a spectrophotometer (1 OD600 = 2×107 cells) at the indicated concentrations of itraconazole (left panel) or 25-thialanosterol (right panel). Error bars represent standard deviation from three biological replicate experiments. B. Wild-type, sre1Δ and stp1Δ cells were grown for 48 hours in liquid YES medium. Equal numbers of cells were plated and grown on YES medium for 2 days. Colony-forming units were counted and percent viability calculated. C. Wild-type, sre1Δ and stp1Δ cells were grown for the indicated times in the presence of 2.5 nM itraconazole, 5 nM 25-thialanosterol or DMSO as a vehicle control. Equal numbers of cells were plated and grown on YES medium for 2 days. Error bars represent the standard deviation from three biological replicate experiments.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.
5.
Figure 2

Figure 2. Sre1 cleavage occurs within the first transmembrane segment. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. Diagram of Sre1 in the membrane. TM1 indicates predicted first transmembrane segment. Amino acid positions are given and bHLH denotes the N-terminal basic helix-loop-helix leucine zipper DNA binding domain. B. Protein sequence alignment of the predicted first transmembrane segments of C. neoformans and human SREBP-2. Putative helix-destabilizing residues are underlined. C. Western blot of phosphatase-treated whole cell extracts (40 µg) from wild-type and stp1Δ cells transformed with empty vector, or plasmids expressing the indicated Sre1 truncations. Lanes 1 and 7, 2 and 6, 3 and 5 are duplicates of stp1Δ cells expressing empty vector, Sre1 (aa1-535), and Sre1 (aa1-501), respectively. Cells were grown to exponential phase in YES medium at ambient oxygen concentrations. Asterisks indicate non-specific, cross-reacting proteins detected by anti-Sre1 antiserum.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.
6.
Figure 1

Figure 1. C. neoformans Stp1 is a conserved Site-2 protease required for Sre1 activation. From: C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs.

A. Protein alignment of human Site-2 protease (NCBI refseq ID: NP_056968) and C. neoformans Stp1 (BROAD ID: CNAG_05742.2) using SIM alignment tool (http://ca.expasy.org/tools/sim.html). Putative catalytic residues are underlined. B. C. neoformans serotype A wild-type and stp1Δ cells were shifted from 21% oxygen to 3% oxygen for indicated periods of time. Immunoblot analysis was performed on whole cell extracts (40 µg) using anti-Sre1 antiserum. P and N denote the precursor and nuclear forms, respectively. Asterisks indicate non-specific, cross-reacting proteins detected by anti-Sre1 antiserum. C. C. neoformans cells from the indicated strains were grown under ambient (21%) or 3% oxygen for 2 hours, and RNA was harvested. SRE1 transcript levels were quantified by real time RT-PCR and normalized to that in wild-type cells at 21% oxygen. Error bars represent standard deviation from three biological replicate experiments. D. Five-fold serial dilutions of C. neoformans cells from the indicated strains were spotted on rich medium and rich medium containing 0.3 mM CoCl2 and incubated at 30°C for 3 days. E. C. neoformans stp1Δ cells transformed with either empty vector, STP1, STP1 (H202A), STP1 (H202A, E203A) and STP1 (D467A) were grown for 2 hours at 3% oxygen. Immunoblot analysis was performed on whole cell extracts (40 µg) using anti-Sre1 antiserum. P and N denote the precursor and nuclear forms, respectively. Asterisk indicates non-specific, cross-reacting proteins detected by anti-Sre1 antiserum. F. C. neoformans stp1Δ cells were transformed with the indicated plasmids and five-fold serial dilutions of cells were spotted on rich medium and rich medium containing 0.3 mM CoCl2 and incubated at 30°C for 3 days. Six independent isolates were plated.

Clara M. Bien, et al. Mol Microbiol. ;74(3):672-690.

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