However, flocculation in response to FeSO4 was less pronounced at that iron concentration compared to 30 μM FeCl3 as quantified by measuring sedimentation rates (Figure 1B) as previously described [33]. Figure 1 Iron induced concentration dependent flocculation of C. albicans cells. (A) Microscopic Milciclib ic50 analysis. C. albicans SC5314 (WT) was incubated with different FeCl3 concentrations (indicated at the top left hand of each sub panel) or with 30 μM FeSO4 in RPMI at 30°C for 2 h. (B) check details Relative sedimentation rates of WT cells. Flocculation of cells was triggered

by 30 μM FeCl3 or 30 μM FeSO4 in RPMI and sedimentation rates were determined after incubation at 30°C for 2 h. Means and standard deviations of three independent samples are shown (n = 3). ** denotes P < 0.01 (student’s t-test). (C) Relative sedimentation rates of WT cells pre-cultured in the sufficient iron (YPD) or restricted iron medium (RIM) at 30°C for 3 h. Flocculation of cells was triggered by 30 μM FeCl3 in RPMI and sedimentation rates were determined after incubation at 30°C for 2 h.

Means and standard deviations of three independent samples are shown (n = 3). *** denotes P < 0.001 (student’s t-test). (D) Microscopic analysis of cycloheximide (CHX) or MeOH pre-treated cells. C. albicans SC5314 was pre-treated either with 500 μg ml-1 CHX or MeOH in RPMI at 30°C for 15 min. Iron or water were subsequently added and cells Vactosertib molecular weight were incubated at 30°C for 2 h. Flocculation was also induced in yeast nitrogen base (YNB) medium containing 30 μM FeCl3 compared to 1.2 μM basal Fe3+ concentration (information given by the manufacturer), thus showing that the induction of flocculation was independent from the medium used (see Additional file 1). Cells may possess internal iron stores from pre-cultivation in an iron sufficient medium. Thus, for we investigated whether the iron content of the medium used during pre-cultivations influenced

the dependence of the flocculent phenotype on the iron concentration in RPMI. C. albicans was either pre-cultivated in a medium with sufficient iron, i.e. the rich yeast extract-peptone-dextrose (YPD) medium, or starved for iron by pre-cultivation in a medium with restricted iron availability (restricted iron medium: RIM). RIM resulted from addition of the iron chelator bathophenanthroline disulfonate (BPS) to YPD medium. As shown in Figure 1C, flocculation due to exposure to 30 μM Fe3+ was independent on the pre-cultivation medium: WT cells starved for iron by pre-cultivation in RIM flocculated upon exposure to 30 μM Fe3+ with a similar sedimentation rate as cells pre-cultivated in YPD. During all later experiments, we pre-cultivated C. albicans in YPD and added 30 μM FeCl3 as iron source to the respective medium of the working culture unless it is mentioned otherwise.