Fig 2. Incorporation of radio-labelled or ¹³C-labelled carbon sources into lipids of PCF trypanosomes.

In panels A and B, [¹⁴C]-labelled fatty acid methyl esters and sterols were separated by HPTLC and analyzed as described in the Materials and methods section. Prior to lipid extraction, the EATRO1125.T7T PCF were incubated 16 h in SDM79 containing 4 mM glucose, 4 mM threonine, 4 mM proline, 4 mM leucine, 1 mM isoleucine, 1 mM valine, 4 mM acetate and one radio-labelled tracer (A, [1-¹⁴C]-acetate; T, L-[U-¹⁴C]-threonine; G, D-[U-¹⁴C]-glucose; P, L-[U-¹⁴C]-proline; L, L-[U-¹⁴C]-leucine; I, L-[U-¹⁴C]-isoleucine; V, L-[U-¹⁴C]-valine). The cell has also been incubated with L-[U-¹⁴C]-proline, 0.15 mM threonine, 1 mM isoleucine and 1 mM valine, without glucose and acetate (P*). The data are expressed as nmol of precursor (radioactive and non-radioactive molecules) incorporated into fatty acids (A) and/or sterols (B) in 10⁸ cells per hour. Error bars indicate mean ± SD of 3 biological replicates. nd: not detectable. In panel C, the EATRO1125.T7T PCF were incubated in the same conditions as described above with non-enriched (control) or uniformly [¹³C]-enriched proline, leucine, glucose, threonine or acetate, before processing the sample for sterol analysis by GC-MS. The isotope enrichment factor corresponds to the percentage of molecules containing ¹³C-enriched carbons. The amount of each of the five sterols identified is similar in the six experimental conditions; cholesta-5,7,24-trienol, 0.5 ± 0.1 μg; prothothecasterol, 2.2 ± 0.6 μg; ergosta-5,7,24(25)-trienol, 0.7 ± 0.1 μg; ergosta-5,7,25(27)-trienol, 2.2 ± 0.4 μg; cholesterol, 7.9 ± 1.7 μg.