These were recorded with the Luminescent Image Analyzer and analyzed by ImageGauge 3. 46 and L Process v 1. 96. Flocculation assay by low speed centrifugation The cells of selleck screening library strains were streaked on YPD agar plate for 3 days and colonies were picked and inoculated into SD medium with required supplements for 48 hrs. Next, the cultures were diluted into fresh SD medium to 0. 1 of an initial OD600 with required supplements. To simultan eously repress the expression of CaMET3p driven CaCDC4 and to induce the expression of various CaCDC4 segments encoding series of CaCdc4 domains, 2. 5 mM Met Cys and 40 ug ml Dox were also added into the SD medium. After 48 hrs, the cultures were spun down for 1 minute at 500 rpm, and the suspensions of the cultures were sampled to determine their optical density at OD600.

Three independent assays were conducted and each sam ple was assayed in duplication. A paired Student t test with p 0. 05 was Batimastat considered significance. Ca2 initiated flocculation assay The FLO encoded flocculins are known to be essential for flocculation in S. cerevisiae. Functional homologues of FLO genes have been found in C. albicans. In particular, the important S. cerevisiae gene FLO11 responsible for flocculation has C. albicans functional counterpart ALS1. Since FLO11 associated flocculation is dependent on the presence of Ca2, we adopted an alternative floccula tion assay in which the rate of flocculation is initiated by Ca2 and the optical density was assessed within a short time frame.

Briefly, to initiate flocculation, an aliquot of 800 ul deflocculated cell suspension was transferred into a 1 ml cuvette, followed by addition of 200 ul of 100 mM CaCl2. The cuvette was mixed robustly by pipet ting and the absorbance was assessed instantly at 30 s intervals for 5 minutes using a spectrophotometer. All assays were con ducted in triplicate. Constructing a C. albicans strain capable add to favorites of conditionally repressing the expression of CaCDC4 To establish C. albicans strains capable of expressing CaCDC4 and its domains solely controlled under a Tet promoter directly in C. albicans, BWP17, with both alleles of CaCDC4 deleted, was constructed to accommodate Tet on plasmid cassettes capable of expressing assorted CaCdc4 domains induced by Dox. The first allele of CaCDC4 was deleted in BWP17 by mini Ura blaster to generate the JSCA0018 strain. This strain was used to delete the second CaCDC4 allele to ob tain a Cacdc4 null mutant. However, Cacdc4 null mutant cells growing as filamentous form with toughened cell walls obstructed transformation. To overcome this problem, the strain JSCA0021 was created that had one CaCDC4 al lele deleted and the other under CaMET3 control that was Met Cys repressible.