Figure 8.

Bloodstream forms without EIF4E1 cannot differentiate into growing procyclic forms. (A) Depletion of EIF4E1 in Lister 427 bloodstream forms results in a very mild growth defect. Cumulative growth of cultures with tetracycline-inducible RNAi is shown, with mean and standard deviation for three independent experiments. (B) Cumulative growth of different EATRO1125 cell lines. For WT and SKO (single replacement), the division times of the same cell lines were measured in two independent experiments. For DKO (both genes replaced), results for three different lines are shown. (C) Effect of adding cis-aconitate (CA) and shifting the temperature to 27°C (EATRO1125). The starting cell density was 1 × 10⁶/ml. EP procyclin expression was measured by western blotting, with ribosomal protein S9 as the loading control. Although it is not evident from the image, the last three lanes are somewhat under-loaded, and quantitation revealed no significant differences between DKO lines and the SKO or WT. (D) Cell numbers in cultures that were treated as in (C) (HMI-9 +CA, 27°C), then transferred to medium suitable for procyclic forms (MEM, 27°C). The results shown are mean and standard deviation for three EIF4E1 cultures. Controls included two wild-type cultures (mean shown) and a 4EIP culture. (E) Expression of PAD1 after incubation at high density. Cells were incubated until they attained 3 × 10⁶/ml, then the first samples were taken. To ensure complete comparability, subsequent samples were taken when the cell numbers had dropped to precisely 1.5 × 10⁶ and 1.0 × 10⁶/ml. Amounts of PAD1 relative to the Ponceau red stain are indicated. (F) Cells were left at high density for 48 h, then treated as in (D). Cumulative cell counts are shown.