Figure 3. High iron conditions result in higher intracellular iron concentrations and sphingolipid LCBs.

A. Intracellular metal content analysis. Strains were grown on SD plates containing a normal concentration of Fe and incubated for 2 days, or on SD+7mM FeCl₃ and incubated for 6 days (pORM2) and 11 days (YEp24) before harvesting for ICP-MS measurement. The results shown are the average of 6 samples (triplicate samples from 2 independent experiments), except for pORM2 in normal conditions, which is the average of 5 samples (3 and 2 samples from 2 independent experiments).

B. Sphingolipid analysis in normal, high Fe and high Cu conditions. Strains were grown either on normal medium, 7mM FeCl₃, 200ng/ml myriocin, 7mMFeCl₃+200ng/ml myriocin or 2mM CuSO₄ conditions and incubated at 25°C until colonies reached a similar size before harvesting for LCBs analysis. Triplicate samples for LCBs analysis were prepared as described in experimental procedures. LCB and LCBP levels are indicated as pmol/A600. Statistical analysis is provided in Supplementary Table S1. Evidence for reproducibility of the effect of high iron conditions and ORM2 overexpression on LCB levels is shown in Figure S3.

C. Comparison of the effects of Orm1p and Orm2p overexpression on sphingolipid levels. Procedures were the same as in B, except that cells were transformed with the vector alone (YEp24), or the vector overexpressing Orm1p or Orm2p (pORM1/2) and grown in -URA medium (lanes 1,2, 3) or with -URA + 7mM FeCl₃. Shown are the average values of 3 independent biological samples with the standard deviations.