This last observation was

also confirmed by co-injecting the extracted sterols with standard ergosterol, resulting in three peaks at approximately Akt inhibitor 15, 18 and 22 min (Figure  6 D). Additionally, the identity of the sterols was determined by GC-MS (Additional file 1: Figure S1), confirming that wild-type strains produced mainly this website Ergosterol and that the mutants instead accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Considering the relative abundance of each sterol obtained by GC-MS and RP-HPLC, peaks 2 and 3 in the RP-HPLC chromatogram from the cyp61 – mutant strain (Figure  6 C) should correspond to ergosta-5,8-dien-3-ol and ergosta-5,8,22-trien-3-ol, respectively. Figure 6 RP-HPLC sterols analysis from UCD 67–385 and 385-cyp61 hph /cyp61 zeo strains. Chromatograms (at 280 nm) correspond Nutlin-3 in vivo to sterols extracted from strains as described in the Materials and Methods section. dendrorhous mutant strains (in mg/g dry yeast weight)   Strains   UCD 67-385 385-cyp61 (+/−) 385-cyp61 (−/−) Cultivation time (h) 24 72 120 24 72 120 24 72 120 Ergosterol* 4.74±0.53 3.10±0.09 2.24±0.42 3.19±0.48 2.87±0.32 2.91±0.34

MTMR9 ND ND ND Peak 2** 0.23±0.03 0.030±0.003 0.10±0.05 0.62±0.05 0.11±0.03 0.12±0.02 6.34±2.68 2.36±0.74 2.39±0.27 Peak 3*** 0.19±0.04 ND 0.09±0.02 0.11±0.01 0.02±0.01 0.01±0.003 1.65±0.84 1.91±0.51 2.20±0.42 Total Sterols 5.16±0.57 3.13±0.09 2.40±0.49 3.96±0.44 2.99±0.35 3.04±0.36 8.14±3.42 4.27±1.24 4.59±0.70   Strains         CBS 6938 CBS – cyp61 (−)       Cultivation time (h) 24 72 120 24 72 120       Ergosterol* 3.31±0.60 2.39±0.56 2.37±0.11 ND ND ND       Peak 2** 0.07±0.04 0.06±0.02 0.06±0.01 2.00±0.34 1.24±0.02 1.23±0.04       Peak 3*** 0.03±0.001 0.02±0.01 0.03±0.01 2.38±0.29 2.60±0.08 3.05±0.17       Total Sterols 3.45±0.56 2.41±0.59 2.46±0.11 4.38±0.61 3.85±0.1 4.28±0.21         Strains         AVHN2 AV2 – cyp61 (−)       Cultivation time (h) 24 72 120 24 72 120       Ergosterol* 1.59±0.62 2.35±0.59 3.27±0.38 ND ND ND       Peak 2** ND 0.04±0.01 0.04±0.01 1.68±0.78 2.10±0.32 1.78±0.13       Peak 3*** ND ND ND 1.39±0.82 2.27±0.18 2.39±0.52       Total Sterols 1.59±0.62 2.39±0.59 3.31±0.39 3.16±1.70 4.36±0.49 4.11±0.64       Table shows the mean values ± standard deviations of three independent experiments.