Skip to main content
. 2018 Aug 16;46(17):8993–9010. doi: 10.1093/nar/gky733

Figure 6.

Figure 6.

Cells without 4EIP differentiate poorly into stumpy forms. (A) Depletion of 4EIP in monomorphic bloodstream forms (Lister 427) results in a very mild growth defect. Cumulative growth of cultures with tetracycline-inducible RNAi is shown, with mean and standard deviation for three different cell lines. Expression of V5-4EIP in one of the three different RNAi lines, analyzed by western blotting, is shown below. TR is trypanothione reductase. Results for the other two cell lines are in Supplementary Figure S7A. (B) Division times of different EATRO1125 cell lines. The division times of the same cell lines were measured in two independent experiments. Results for the individual replicates are shown. KO: no 4EIP; K+: knockout line with inducible 4EIP-myc, with or without tetracycline; ΔN—knockout line with inducible lambdaN-ΔN-4EIP-myc, with or without tetracycline. Tetracycline was added 48 h prior to the start of measurements. In the second experiment (diamonds), the knockout line appeared to have adapted to the lack of 4EIP during continuous culture. (C) Growth of different EATRO1125 cell lines allowed to reach high density. Cells with a starting density of 5 × 105/ml were allowed to grow in HMI-9 media containing 1.1% methylcellulose for 60 h. Results are mean and standard deviation of three replicates. K+ are the complemented cells and K± are the K+ cells grown without tetracycline. (D) [35S]-Methionine incorporation into proteins during differentiation of EATRO1125 at high density. Trypanosomes were cultured as shown in (B), washed and pre-incubated for 1 h in labeling medium at 37°C. Methionine (35S) was then added for 20 min and proteins were analyzed by SDS-PAGE (Supplementary Figure S8B). Results for seven independent experiments are shown; four of the experiments included time = 0 h and three included time = 60 h. The color code is a paler version of (B). Student’s t-test results are: ** P < 0.01, * P < 0.05, both as paired or unpaired tests. Expression of various proteins, by western blotting, is shown under the box plot. For 4EIP-myc, the full-length protein is shown; degradation products are shown in Supplementary Figure S6F. The PAD1 signals from WT and K± are comparable.