Figure 2. Glutamine-derived carbons are incorporated into amastigote sterols and influence the buildup of lanosterol.

(A) Chromatogram from GC-MS detection of samples. Host-cell-derived cholesterol is seen at retention time 12.21, eburicol at 13.30, lanosterol at 13.04 and the internal standard at 12.98. (B) Table of detectable isolated amastigote sterol species from panel A and the percentage of natural sterols (i.e. without detectable ¹³C). The proportion of species found with the indicated number of incorporated ¹³C carbons are shown (e.g. 13C1, 13C2). (C) Quantification, using an internal standard, of lanosterol and (D) eburicol in isolated amastigotes (52 hpi) following treatment with ketoconazole (5 nM) at 18 hpi with or without glutamine (2 mM). Mean and standard deviation shown of independent treatments, infections and amastigote isolations (n = 2). Statistical comparisons are made using a Student’s t-test (*p<0.05, ns = not significant).

Figure 2—figure supplement 1. Time course establishes 52 hpi as optimal time point to harvest intracellular amastigotes following ketoconazole treatment.

Time course following treatment with ketoconazole (5 nM) in complete media. (A) Amastigotes per infected host cell (n = 40) and (B) infected cells per 20 fields are shown. Fifty-two hpi identified as maximum time of ketoconazole exposure prior to measurable loss of intracellular amastigotes.

Figure 2—figure supplement 2. Endogenous lanosterol and eburicol but not host derived cholesterol are reliable quantifiable from isolated intracellular amastigotes.

Isolated amastigotes (52 hpi) were prepared on three independent occasions (biological) and each isolation was extracted three separate times (sterol extraction). The coefficient of variation of standard normalized area (GC-MS) was determined, means indicated. Variation in cholesterol between biological replicates prohibits reliable quantification.